Upload
kitkat-alorro
View
218
Download
0
Embed Size (px)
Citation preview
7/31/2019 Bio 108 Lec 5
1/68
DNA REPLICATION-in all cells, DNA sequences should be maintained and replicatedwith high fidelity (mutation rate, approximately 1 nucleotidechange per 109 nucleotides each time the DNA is replicated, isroughly the same for organisms as different as bacteria andhumans).-the sequence of the human genome (approximately 3 109
nucleotide pairs) is changed by only about 3 nucleotides each time acell divides.-this allows most humans to pass accurate genetic instructions fromone generation to the next, and also to avoid the changes in somaticcells that lead to cancer
Why Study DNA Replication?
1) Understanding Cancer-- the uncontrolled cell division (DNA replication).2) Understanding Aging--cells are capable of a finite number of doublings.
3) Understanding Diseases associated with defects in DNA repair.
DNA REPLICATION, REPAIR and RECOMBINATION
7/31/2019 Bio 108 Lec 5
2/68
1) CancerCells are carefully controlled in the number of cell doublings that they arecapable of as well as when cell division will occur. In cancer the control ofinitiation of replication is lost
2) AgingFor example, fibroblast cells (connective tissue) in culture will double forabout 50 generations. Then they enter senescence. Senescent cells are nolonger capable of dividing yet remain metabolically active. In addition,they exhibit changes in form and function, which may lead to age-related
changes such as the difference between the supple skin of a child and thewrinkled skin of the elderly.3) DNA repair diseases
There are several diseases that cause premature aging or sensitivity to UVlight.
Examples include:
a) Bloom Syndrome, a cancer-prone genetic disorder due to genetic instability inthe form of increased frequencies of breaks of the chromosomes.
b) Xeroderma Pigmentosum, a human DNA repair deficiency syndrome leadingto predisposition to sun-light-induced skin cancer.
c)Werner Syndrome, a premature aging disease that begins in adolescence orearly adulthood and results in the appearance of old age by 30-40 years of
age.
7/31/2019 Bio 108 Lec 5
3/68
Xeroderma patient
7/31/2019 Bio 108 Lec 5
4/68
7/31/2019 Bio 108 Lec 5
5/68
KEY CONCEPTS:
Proteins interact with DNA in all biological activities involvingDNA.
DNA must be unwound to replicate. Topoisomerases catalyze changes in supercoiled state of DNA. DNA replication has three distinct phases (initiation, elongation,
and termination). Termination is different at telomeres ofeucaryotic chromosomes
DNA replication is very accurate (1x10-8 mistakes/base).DNA molecules can recombine if they have similar sequences. Mutations have several causes and involve base sequence changes.
DNA repair corrects errors using highly evolved correctionsystems.
7/31/2019 Bio 108 Lec 5
6/68
Three general features of Chromosomal replication:1. DNA Replication Is Semiconservative
1958: Meselson and Stahl: DNA
Replication is Semiconservative
*(in both prokaryotes & eukaryotes)
7/31/2019 Bio 108 Lec 5
7/68
2. Most DNA Replication Is Bidirectional
Figure 12-2. Three mechanisms of DNA strand growth that are consistent with
semiconservative replication.The third mechanismbidirectional growth of both strandfrom a single originappears to be the most common in both eukaryotes and prokaryotes.
7/31/2019 Bio 108 Lec 5
8/68
Because of the anti-parallel structure of the DNA duplex, new DNA must besynthesized in both the 5 to 3 and 3 to 5 directions overall.However all known DNA polymerases synthesize DNA in the 5 to 3 directiononly.
The solution is semidiscontinuous DNA replication.
Leading Strand-replicates continuously
Lagging Strand-replicates discontinuously-consists of OkasakiFragments (ss DNA chains1000-2000 nucleotides long,primed by very short RNA
primers) which need to bejoined by DNA ligase-the parental strand forms atrombone structure
RNA primers
The Leading and Lagging
Strands Are Synthesized
Concurrently
7/31/2019 Bio 108 Lec 5
9/68
7/31/2019 Bio 108 Lec 5
10/68
3. DNA Replication Begins at Specific Chromosomal Sites-DNA synthesis is initiated at special regions called replication origins. Abacterial chromosome has one origin, whereas each eukaryotic
chromosome has many (hundreds or even thousands).Close-up of a replication forkorigin of replication
Bubble
Parental (template) strandDaughter (new) strand
Replication fork
Two daughter DNA molecules
7/31/2019 Bio 108 Lec 5
11/68
Figure 5.14. Origin of replication inE. coli Replication initiates
at a unique site on the E. coli chromosome, designated theorigin (ori)
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.28867/31/2019 Bio 108 Lec 5
12/68
Figure 5.15. Replication origins in eukaryotic chromosomes Replicationinitiates at multiple origins (ori), each of which produces two replication
forks.
7/31/2019 Bio 108 Lec 5
13/68
Three Common Features of Replication Origins
1. replication origins are unique DNA segments that contain multiple
short repeated sequences
2. these short repeat units are recognized by multimeric origin-binding
proteins.
3. origin regions usually contain an AT-rich stretch
*Origin-binding proteins control the initiation of DNA replication bydirecting assembly of the replication machinery to specific sites on theDNA chromosome.
Replicon - region of DNA served by one replication origin.
Three types of replication origins:1. E. coli oriC2. yeast autonomously replicating sequences (ARS)3. simian virus 40 (SV40) origin.
7/31/2019 Bio 108 Lec 5
14/68
1. oriCis an 240-bp DNA segment present at the start site for replicationof E. coli chromosomal DNA-contain repetitive 9-bp and AT-rich 13-bp sequences, referred toas 9-mers (dnaA boxes) and 13-mers, respectively.
Figure 12-5. Consensus sequence of the minimalbacterial replication origin
*these are binding sites for the DnaA protein that initiatesreplication.
7/31/2019 Bio 108 Lec 5
15/68
2. Yeast Autonomously Replicating Sequences- has multiple origins of replication-confers on a plasmid the ability to replicate in yeast and is arequired element in yeast artificial chromosomes-a 15-bp segment, designated element A, stretching fromposition 114 through 128 which contains an 11-base-pair ARSconsensus sequence (ACS), which is the specific binding site ofthe origin replication complex (ORC).-three additional elements (B1, B2, and B3) are individually not
essential but together contribute to ARS function.
Figure 5.17. A yeastARS element
7/31/2019 Bio 108 Lec 5
16/68
3. SV40 Replication Origin-A 65-bp region in the SV40 chromosome is sufficient to promoteDNA replication both in animal cells and in vitro.
-three segments of the SV40 origins are required for activity-initiated by a virus-encoded protein (called T antigen) that bindsto the origin and also acts as a helicase.
The DNA Replication MachineryDNA Polymerases
DNA polymerases are unable to melt duplex DNA (i.e., break theinterchain hydrogen bonds) in order to separate the two strands that are tobe copied.
All DNA polymerases so far discovered can only elongate apreexisting DNA or RNA strand, the primer; they cannot initiate chains.
The two strands in the DNA duplex are opposite (53 and 35)in chemical polarity, but all DNA polymerases catalyze nucleotide additionat the 3-hydroxyl end of a growing chain, so strands can grow only in the53 direction.
*In this section, we describe the cell's solutions to the unwinding,priming, and directionality problems resulting from the structure of DNAand the properties of DNA polymerases
T bl 2 S f h P i R i d f R li i
7/31/2019 Bio 108 Lec 5
17/68
E. coliprotein Eukaryotic protein Function
DnaA ORC proteins Recognition of origin of replication
Gyrase Topoisomerase I/II Relieves positive supercoils ahead of
replication fork
DnaB Mcm DNA helicase that unwinds parentalduplex
DnaC ? Loads helicase onto DNA
SSB RFA Maintains DNA in single-stranded state
-complex RFC Subunits of the DNA polymerase
holoenzyme that load the clamp onto
the DNApol III core pol / Primary replicating enzyme; synthesizes
entire leading strand and Okazaki
fragments; has proofreading capability
subunit PCNA Ring-shaped subunit of DNA polymerase
holoenzzyme that clamps replicating
polymerase to DNA;works with pol III in
E. coli and pol or in eukaryotes
Primase Primase Synthesizes RNA primers
- pol Synthesizes short DNA oligonucleotides
as part of RNA-DNA primer
DNA ligase DNA ligase Seals Okazaki fragments into continuous
strandpol I FEN-1 Removes RNA primers; pol I ofE.coli
Table 2. Some of the Proteins Required for Replication
7/31/2019 Bio 108 Lec 5
18/68
Figure 12-7. Model of initiation ofreplication atE. coli oriC.
DnaA Protein Initiates
Replication in E. coli
DnaB Is an E. coliHelicase That
Melts Duplex DNA
7/31/2019 Bio 108 Lec 5
19/68
Replication overview
Must maintain integrity of the DNA sequencethrough successive rounds of replication
Need to:
unwind DNA, add an RNA primer, find anappropriate base, add it to the growing DNAfragment, proofread, remove the initial primer,fill in the gap with DNA, ligate fragmentstogether
All of this is fast, about 100 bp/second
7/31/2019 Bio 108 Lec 5
20/68
Table 12 1 Properties of DNA Polymerases
7/31/2019 Bio 108 Lec 5
21/68
Table 12-1. Properties of DNA PolymerasesE. coli I II III
Polymerization:
53
+ + +
Exonuclease activity:
35 + + +
53 +
Synthesis from:
Intact DNA Primed single
strands
+
Primed single
strands plus single-
strand-binding
protein
+ +
In vitro chain
elongation rate
(nucleotides per
minute)
600 ? 30,000
Molecules present
per cell
400 ? 1020
Mutation lethal? + +
7/31/2019 Bio 108 Lec 5
22/68
Mammalian Cells*
Polymerization:
53
+ + + + +
Exonuclease
proofreading
activity:3 5
+ + +
Synthesis from:
RNA primer + + ?
DNA primer + + + + +
Associated DNA
primase
+
Sensitive to
aphidicolin (inhibitor
of cell DNA
synthesis)
+ + +
Cell location:
Nuclei + + + +Mitochondria +
*Yeast DNA polymerase I, II, and III are equivalent to polymerase,, and, respectively. I and III are essential for cell viability.Polymerase is most active on DNA molecules with gaps of about 20 nucleotides and is thought to play a role in DNA repair.FEN1 is the eukaryotic 53exonuclease that removes RNA primers; it is similar in structure and function to the domain ofE.
colipolymerase I that contains the 53exonuclease activity.
7/31/2019 Bio 108 Lec 5
23/68
The first DNA polymerase was discovered by Arthur Kornberg in 1957: Pol I
E. coli DNA Pol I has 3 enzymatic activities:
1) Polymerization 5 32) Exonuclease 3 5 (Proofreading)3) Exonuclease 5 3 (Edit out sections of damaged DNA)
Klenow Fragment
DNA Polymerase Error Rate = 1/ 109 bp = 1 X 109 in the cell
100-1000X better than RNA Polymerase
DNA Pol III is highly processive while DNA Pol I is distributive
Processivity is continuous synthesis by polymerase without dissociation fromthe template.
A DNA polymerase that is Distributive will dissociate from the template aftereach nucleotide addition
Pol I & II main DNA repair enzymePol III main DNA replication enzyme
7/31/2019 Bio 108 Lec 5
24/68
Helicase -unwinds DNA. (ATP hydrolysis required -
introduces positive supercoils.)SSB protein (single-strand-binding protein) -binds to
the parental single strands as they are unwoundto prevent reannealing.
DNA gyrase -introduces negative supercoils to relievetorsional strain (ATP hydrolysis required).
RNA primase- (a specific RNA polymerase)synthesizes a primer of about 5bases long. TheRNA primer is later removed (and the gap filled
in) by Pol I.Pol III dimer-adds deoxyribonucleotides to the RNA
primer.
LEADING STRAND SYNTHESIS (elongation)
primosomeis now generally used to denote a complex between
primase and helicase, sometimes with other accessory proteins.
7/31/2019 Bio 108 Lec 5
25/68
Figure 5.11. Model of theE. colireplication fork
Model for the replication machine, or replisome
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.28867/31/2019 Bio 108 Lec 5
26/68
7/31/2019 Bio 108 Lec 5
27/68
Eukaryotic Replication Machinery Is Generally Similar to That ofE. coli
(refer to Table 2 for the proteins used)
TERMINATION OF DNA REPLICATION :-Pol I cleaves off RNA primers and fills in gaps (both
leading and lagging strands); as well as Rnase H(bacteria)
-DNA ligase seals gaps.
Figure 7-2. Plasmid DNA replication
7/31/2019 Bio 108 Lec 5
28/68
Telomerase Prevents Progressive Shortening of Lagging Strands duringEukaryotic DNA Replication
Termination of Eucaryotic DNA replication: The Problem - its a linearchromosome, so how to complete the ends?? (Cant just ligate ends and get a
circle as with E. coli chromosome;
Eucaryotic
Telomere structure
Telomerase
Ends of linear DNA will be
shortened by replication
Lagging strand cannot be primed beyond end of
leading strand, but the leading strand is
shortened due to priming.
Therefore, chromosomal end must be repaired
Telomerase is an RNA-directed DNApolymerase, containing RNA template.
7/31/2019 Bio 108 Lec 5
29/68
DNA replication leaves one incomplete end Telomere synthesis by telomerase
7/31/2019 Bio 108 Lec 5
30/68
Telomerase and Cancer
Germ cells and rapidly dividing somatic cells produce
telomerase.
Most human somatic cells lack telomerase, leading toshortening of telomeres with cell division.
Most tumor cells express telomerase.
Telomerase knockout mice are viable (!), but less able to
produce tumors.
Telomerase inhibitors may be valuablechemotherapeutics (e.g., Gerons GRN163L started
clinical trials for breast cancer August, 2008).
Telomerase activators may be valuable for regeneration
(e.g. Gerons TAT2 increases telomerase activity andproliferative capacity in cytotoxic T-cells in HIV-infected
pts.)
7/31/2019 Bio 108 Lec 5
31/68
DNA TOPOLOGY: DNA-BINDING PROTEINS ALTER THE TOPOLOGY OF DNA
Negative supercoiled circular DNA is compact and is energetically favored.
Most DNA in cells has negative supercoiled (right-handed)
superhelices.Superhelices are underwound. This facilitated DNA
helix unwinding for replication, recombination, transcription, etc.
Positive supercoils(left-handed) make opening the helix more difficult.
The topology of DNA (state of supercoiling) can be changed by unwinding or
winding supercoils. Changes in linking number result in different DNAtopoisomers. Changes require cutting one or both DNA strands.
7/31/2019 Bio 108 Lec 5
32/68
Different states of DNA supercoiling (negative and positive)
Topoisomerases,enzymes that catalyze the reversible breakage and
rejoining of DNA strands
Topoisomerase enzymes can DNA convert + to - supercoils
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.28867/31/2019 Bio 108 Lec 5
33/68
Type I topoisomerases relax DNA (i.e., remove supercoils) by nicking and
closing one strand of duplex DNA
Topoisomerase I
[1 strand cut]
[left-handed supercoils]
Type II topoisomerases change DNA topology by breaking and rejoining
double-stranded DNA. These enzymes can introduce or remove supercoils and
can separate two DNA duplexes that are intertwined
Topoisomerase II[2 strands cut]
[right-handed supercoils]
(DNA Gyrase - uses ATP)
*Two DNA gyrase inhibitors arenalidixic acid (prevents strand
cutting and rejoining) and
novobiocin (blocks ATP binding) are.
Both replicated circular and linear DNA chromosomes are separatedby type II topoisomerases.
(NOTE: Helicase in DNA replication adds positive
supercoils, makes NO cuts, and uses ATP)
7/31/2019 Bio 108 Lec 5
34/68
The Role of Topoisomerases in DNA Replication
Figure 12-14. Action ofE. coli type Itopoisomerase (Topo I). The DNA-
enzyme intermediate contains acovalent bond between the 5-phosphoryl end of the nicked DNAand a tyrosine residue in the protein(inset). After the free 3-hydroxyl endof the red cut strand passes under theuncut strand, it attacks the DNA-enzyme phosphoester bond, rejoiningthe DNA strand. During each roundof nicking and resealing catalyzed byE. coli Topo I, one negative supercoilis removed. (The assignment of signto supercoils is by convention withthe helix stood on its end; in anegative supercoil the front strandfalls from right to left as it passes overthe back strand (as here); in a positive
supercoil, the front strand falls fromleft to right.)
7/31/2019 Bio 108 Lec 5
35/68
Figure 12-16. Action ofE. coli
DNA gyrase, a type IItopoisomerase. (a) Introduction
of negative supercoils. The initial
folding introduces no stable
change, but the subsequent
activity of gyrase produces a
stable structure with two
negative supercoils. Eukaryotic
Topo II enzymes cannot introduce
supercoils but can remove
negative supercoils from DNA. (b)
Catenation and decatenation oftwo different DNA duplexes. Both
prokaryotic and eukaryotic Topo
II enzymes can catalyze this
reaction.
7/31/2019 Bio 108 Lec 5
36/68
Fidelity of DNA replication can be traced to three distinct
activities:
1. accurate selection of nucleotides
2. immediate proofreading
3. postreplicative mismatch repair
DNA RepairTo maintain the integrity of their genomes,
cells have therefore had to evolve mechanisms to
repair damaged DNA.
A failure to repair DNA produces a mutation.
The recent publication of the human genome has already
revealed 130 genes whose products participate in DNA repair.
More will probably be identified soon.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mutations.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mutations.html7/31/2019 Bio 108 Lec 5
37/68
Agents that Damage DNA
Certain wavelengths of radiation
ionizing radiation such as gamma rays and x-rays
ultraviolet rays, especially the UV-C rays (~260 nm) that are
absorbed strongly by DNA but also the longer-wavelength UV-B that
penetrates the ozone shie ld
Highly-reactive oxygen radicals produced during normal cellular
respiration as well as by other biochemical pathways.
Chemicals in the environment
many hydrocarbons, including some found in cigarette smoke
some plant and microbial products, e.g. the aflatoxins produced
in moldy peanuts
Chemicals used in chemotherapy, especially chemotherapy of
cancers
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RadiantEnergy.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/U/Units.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/H/Hydrocarbons.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNArepair.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNArepair.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/H/Hydrocarbons.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/U/Units.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RadiantEnergy.html7/31/2019 Bio 108 Lec 5
38/68
Figure 5.20. Examples of DNA
damage induced by radiation and
chemicals (A) UV light induces the
formation of pyrimidine dimers, inwhich two adjacent pyrimidines
(e.g., thymines) are joined by a
cyclobutane ring structure. (B)
Alkylation is the addition of methyl
or ethyl groups to various positions
on the DNA bases. In this example,alkylation of the O6 position of
guanine results in formation of O6-
methylguanine. (C) Many
carcinogens (e.g., benzo-(a)pyrene)
react with DNA bases, resulting in
the addition of large bulky chemicalgroups to the DNA molecule.
7/31/2019 Bio 108 Lec 5
39/68
Types of DNA Damage1.All four of the bases in DNA (A, T, C, G) can be covalently modified at various
positions.
One of the most frequent is the loss of an amino group ("deamination") resulting, for example, in a C being converted to a U.
2.Mismatches of the normal bases because of a failure of proofreading during
DNA replication.
Common example: incorporation of the pyrimidineU (normally found only in
RNA) instead ofT.
3.Breaks in the backbone.
Can be limited to one of the two strands (a single-stranded break, SSB) or
on both strands (a double-stranded break (DSB).
Ionizing radiation is a frequent cause, but some chemicals produce breaks as
well.
4.Crosslinks Covalent linkages can be formed between bases
on the same DNA strand ("intrastrand") or
on the opposite strand ("interstrand").Several chemotherapeutic drugs used against cancers crosslink DNA
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/Groups_5.gifhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAReplication.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAReplication.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/Groups_5.gif7/31/2019 Bio 108 Lec 5
40/68
Figure 5.19. Spontaneous
damage to DNA There are twomajor forms of spontaneous
DNA damage: (A) deamination
of adenine, cytosine, and
guanine, and (B) depurination
(loss of purine bases) resulting
from cleavage of the bond
between the purine bases and
deoxyribose, leaving an
apurinic (AP) site in DNA.
dGMP = deoxyguanosine
monophosphate.
Table 12-2. DNA Lesions That Require Repair
7/31/2019 Bio 108 Lec 5
41/68
DNA Lesion Example/Cause
Missing base Removal of purines by acid and heat (underphysiological conditions 104 purines/day/cell in a
mammalian genome); removal of altered bases
(e.g., uracil) by DNA glycosylases
Altered base Ionizing radiation; alkylating agents (e.g.,
ethylmethane sulfonate)
Incorrect base Mutations affecting 35 exonuclease
proofreading of incorrectly incorporated bases
Bulge due to deletion or insertion of a nucleotide Intercalating agents (e.g., acridines) that cause
addition or loss of a nucleotide during
recombination or replication
Linked pyrimidines Cyclotubyl dimers (usually thymine dimers)
resulting from UV irradiation
Single- or double-strand breaks Breakage of phosphodiester bonds by ionizing
radiation or chemical agents (e.g., bleomycin)
Cross-linked strands Covalent linkage of two strands by bifunctional
alkylating agents (e.g., mitomycin C)
3-deoxyribose fragments Disruption of deoxyribose structure by free radicalsleading to strand breaks
7/31/2019 Bio 108 Lec 5
42/68
These mechanisms of DNA repair can be divided into two general
classes:
(1) direct reversal of the chemical reaction responsible for
DNA damage, and(2) Excision Repair- removal of the damaged bases followed
by their replacement with newly synthesized DNA.
Three types of excision repair
1. BASE-EXCISION REPAIR (BER)2. NUCLEOTIDE-EXCISION REPAIR,(NER)
3. MISMATCH REPAIR (MMR)
Postreplication Repair1. RECOMBINATIONAL REPAIR2. ERROR-PRONE REPAIR.
7/31/2019 Bio 108 Lec 5
43/68
Figure 5.21. Direct
repair of thymine
dimers UV-induced
thymine dimers can
be repaired by
photoreactivation, inwhich energy from
visible light is used to
split the bonds
forming the
cyclobutane ring.
7/31/2019 Bio 108 Lec 5
44/68
Figure 5.22. Repair of
O6-methylguanine
O6-methylguanine
methyltransferase
transfers the methyl
group from O6-
methylguanine to a
cysteine residue in
the enzyme's active
site.
7/31/2019 Bio 108 Lec 5
45/68
Figure 5-50. Acomparison of twomajor DNA repair
pathways.
7/31/2019 Bio 108 Lec 5
46/68
Figure 12-26. Excision repair of DNAbyE. coli UvrABC mechanism. Twomolecules of UvrA and one of UvrBform a complex that moves randomly
along DNA (steps 1 and 2). Once thecomplex encounters a lesion,conformational changes in DNA,powered by ATP hydrolysis, cause thehelix to become locally denatured andkinked by 130 (step 3). After the UvrA
dimer dissociates (step 4), the UvrCendonuclease binds and cuts thedamaged strand at two sites separatedby 12 or 13 bases (steps 5 and 6). UvrBand UvrC then dissociate, and helicaseII unwinds the damaged region (step
7), releasing the single-strandedfragment with the lesion, which isdegraded to mononucleotides. The gapis filled by DNA polymerase I, and theremaining nick is sealed by DNA ligase(step 8). [Adapted from A. Sancar andJ. Hearst, 1993, Science259:1415.]
7/31/2019 Bio 108 Lec 5
47/68
Figure 12-24. Model of mismatch
repair by the E. coliMutHLS system.
This repair system operates soon after
incorporation of a wrong base, before
the newly synthesized daughter strandbecomes methylated. MutH binds
specifically to a hemimethylated GATC
sequence, and MutS binds to the site
of a mismatch. Binding of MutL
protein simultaneously to MutS and to
a nearby MutH activates the
endonuclease activity of MutH, which
then cuts the unmethylated (daughter)
strand in the GATC sequence. A stretch
of the daughter strand containing the
mispaired base is excised, followed by
gap repair and ligation and then
methylation of the daughter strand.
[Adapted from R. Kolodner, 1996,
Genes and Develop.10:1433; see also
A. Sancar and J. Hearst, 1993, Science
259:1415.]
7/31/2019 Bio 108 Lec 5
48/68
Figure 5.25. Mismatch repair in E. coliFigure 5.26. Mismatch repair in
mammalian cells
T bl 5 1 E I l d i N l tid E i i R i
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.28867/31/2019 Bio 108 Lec 5
49/68
Human Yeast Function
XPA RAD14 Damage recognition
XPB RAD25 Helicase
XPC RAD4 DNA binding
XPD RAD3 Helicase
XPF RAD1 5 nuclease
XPG RAD2 3 nuclease
ERCC1 RAD10 Dimer with XPF
Table 5.1. Enzymes Involved in Nucleotide-Excision Repair
Table 5-2. Inherited Syndromes with Defects in DNA Repair
7/31/2019 Bio 108 Lec 5
50/68
NAME PHENOTYPE ENZYME OR PROCESS AFFECTED
MSH2, 3, 6, MLH1, PMS2 colon cancer mismatch repair
Xeroderma pigmentosum (XP)
groups AG
skin cancer, cellular UV sensitivity,
neurological abnormalities
nucleotide excision-repair
XP variant cellular UV sensitivity translesion synthesis by DNA
polymerase
Ataxiatelangiectasia (AT) leukemia, lymphoma, cellular -ray sensitivity, genome instability
ATM protein, a protein kinaseactivated by double-strand breaks
BRCA-2 breast and ovarian cancer repair by homologous
recombination
Werner syndrome premature aging, cancer at several
sites, genome instability
accessory 3-exonuclease and DNA
helicase
Bloom syndrome cancer at several sites, stunted
growth, genome instability
accessory DNA helicase for
replication
Fanconi anemia groups AG congenital abnormalities,
leukemia, genome instability
DNA interstrand cross-link repair
46 BR patient hypersensitivity to DNA-damaging
agents, genome instability
DNA ligase I
7/31/2019 Bio 108 Lec 5
51/68
Figure 5.27. Postreplication repair The
presence of a thymine dimer blocks
replication, but DNA polymerase can bypassthe lesion and reinitiate replication at a
new site downstream of the dimer. The
result is a gap opposite the dimer in the
newly synthesized DNA strand. In
recombinational repair, this gap is filled by
recombination with the undamagedparental strand. Although this leaves a gap
in the previously intact parental strand, the
gap can be filled by the actions of
polymerase and ligase, using the intact
daughter strand as a template. Two intact
DNA molecules are thus formed, and the
remaining thymine dimer eventually can be
removed by excision repair
Repairing Strand Breaks
7/31/2019 Bio 108 Lec 5
52/68
-Ionizing radiation and certain chemicals can produce both:
1. single-strand breaks (SSBs) and
2. double-strand breaks (DSBs) in the DNA backbone.
Single-Strand Breaks (SSBs)
-breaks in a single strand of the DNA molecule are repaired using the same
enzyme systems that are used in Base-Excision Repair (BER).
Double-Strand Breaks (DSBs)-there are two mechanisms by which the cell attempts to repair a completebreak in a DNA molecule:
Direct joiningof the broken ends. This requires proteins that recognize and bind to
the exposed ends and bring them together for ligating. They would prefer to see
some complementary nucleotides but can proceed without them so this type ofjoining is also called Nonhomologous End-Joining (NHEJ).
A protein called Ku is essential for NHEJ. Ku is a heterodimer of the subunits Ku70
and Ku80. In the 9 August 2001 issue ofNature, Walker, J. R., et al, report the
three-dimensional structure of Ku attached to DNA. Their structure shows
beautifully how the protein aligns the broken ends of DNA for rejoining.
Figure 12-28. Repair of double-strand
7/31/2019 Bio 108 Lec 5
53/68
breaks by end-joining of
nonhomologous DNAs (dark and light
blue), that is, DNAs with dissimilar
sequences at their
ends. These DNAs could be cut
fragments from a single gene, or DNAs
cut from different chromosomes. A
complex of two proteins, Ku and DNA-
dependent protein kinase
, binds to the ends of a double-strand
break. After formation of a synapse in
which the broken ends overlap, Kuunwinds the ends, by chance revealing
short homologous sequences in the two
DNAs, which base-pair to yield a region
of microhomology. The unpaired single-
stranded 5 ends are removed by
mechanisms that are not well
understood, and the two double-stranded molecules ligated together. As a
result, the double-strand break is
repaired, but several base pairs at the
site of the break are removed. [Adapted
from G. Chu, 1997,J. Biol. Chem.
272:24097; M. Lieber et al., 1997, Curr.
Opin. Genet. Devel.7:99.]
E i di j i i b f h i l i h
7/31/2019 Bio 108 Lec 5
54/68
Errors in direct joining may be a cause of the various translocations that are
associated with cancers.
Examples:
Burkitt's lymphoma
the Philadelphia chromosome in chronic myelogenous leukemia (CML)B-cell leukemia
Homologous Recombination. Here the broken ends are repaired using the
information on the intactsister chromatid (available in G2 after chromosome duplication), or on the
homologous chromosome (in G1; that is, before each chromosome has been
duplicated). This requires searching around in the nucleus for the homolog a
task sufficiently uncertain that G1 cells usually prefer to mend their DSBs by
NHEJ. or on the
same chromosome if there are duplicate copies of the gene on the
chromosome oriented in opposite directions (head-to-head or back-to-back).
Two of the proteins used in homologous recombination are encoded by the genes
BRCA1 and BRCA2. Inherited mutations in these genes predispose women to breast
and ovarian cancers.
Homologous DNA
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CML.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BCL-2.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Chromosomes.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellCycle.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellCycle.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellCycle.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellCycle.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellCycle.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Chromosomes.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Chromosomes.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BCL-2.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BCL-2.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BCL-2.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CML.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CML.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CML.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CML.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.html7/31/2019 Bio 108 Lec 5
55/68
Homologous DNA
7/31/2019 Bio 108 Lec 5
56/68
Figure 5-53. Two different
types of end-joining for
repairing double-strand
breaks. (A) Nonhomologous
end-joining alters the original
DNA sequence when
repairing broken
chromosomes. These
alterations can be either
deletions (as shown) or short
insertions. (B) Homologous
end-joining is more difficult
to accomplish, but is much
more precise.
7/31/2019 Bio 108 Lec 5
57/68
Inducible DNA-Repair Systems Are Error-Prone
-SOS repair system of bacteriathis system generates many errors in the DNA as it repairs lesions, it
is referred to asrepairs UV-induced damage, differs from the constitutive UvrABC
system
its activity is dependent on RecA protein
errors induced by the SOS system are at the site of lesions,
suggesting that the mechanism of repair is insertion of random
nucleotides in place of the damaged ones in the DNA.
*many investigators believe that in animal cells, as in bacteria,
most mutations are an indirect, not direct, consequence of DNA
damage.
Both bacterial and eukaryotic cells have inducible DNA-repair systems,
which are expressed when DNA damage is so extensive that replication
may occur before constitutive mechanisms can repair all the damage.
7/31/2019 Bio 108 Lec 5
58/68
Figure 8-4. Differenttypes of mutations
7/31/2019 Bio 108 Lec 5
59/68
Recombination- three different functions:
1. increasing genetic diversity which results in the exchange
of genes between paired homologous chromosomes
during meiosis
2.plays
also an important mechanism for repairingdamaged DNA
3. is involved in rearrangements of specific DNA sequences
that alter the expression and function of some genes
during development and differentiation
Thus, recombination plays important roles in the lives of individual cells and
organisms, as well as contributing to the genetic diversity of the species.
R bi ti
7/31/2019 Bio 108 Lec 5
60/68
Recombination
DNA rearrangements are caused by a set of mechanisms that
are collectively called genetic recombination.Two broad classes:
1. general recombination
2. site-specific recombination.
General recombination (also known as homologous recombination)-genetic exchange takes place between a pair of homologous DNAsequences
The breaking and rejoining of two homologous
DNA double helices creates two DNA
molecules that have crossed over. In meiosis,
this process causes each chromosome in a
germ cell to contain a mixture of maternally
and paternally inherited genes.
DNA Molecules Recombine by Breaking and Rejoining
7/31/2019 Bio 108 Lec 5
61/68
Figure 5.28. Models ofrecombination In copy
choice, recombination occurs
during the synthesis of
daughter DNA molecules.
DNA replication starts with
one parental DNA templateand then switches to a
second parental molecule,
resulting in the synthesis of
recombinant daughter DNAs
containing sequences
homologous to both parents.In breakage and rejoining,
recombination occurs as a
result of breakage and
crosswise rejoining of
parental DNA molecules.
DNA Molecules Recombine by Breaking and Rejoining
Holliday model
7/31/2019 Bio 108 Lec 5
62/68
Holliday model
Figure 5.31. The Holliday model
for homologous recombination
Holliday junction The centralintermediate in recombination,consisting of a crossed-strandstructure formed by
homologous base pairingbetween strands of two DNAmoleucles.
Figure 5 33 Isomerization and
7/31/2019 Bio 108 Lec 5
63/68
Figure 5.33. Isomerization and
resolution of Holliday
junctions Holliday junctions
are resolved by cutting and
rejoining of the crossed
strands. If the Holliday
junction formed by the initial
strand exchange is resolved,
the resulting progeny are
heteroduplexes but are not
recombinant for geneticmarkers outside of the
heteroduplex region. Two
rotations of the crossed-strand
molecule, however, produce
an isomer in which the
unbroken parental strands,rather than the initially nicked
strands, are crossed. Cutting
and rejoining of the crossed
strands of this isomer yield
progeny that are recombinant
heteroduplexes.
E I l d i H l R bi i
7/31/2019 Bio 108 Lec 5
64/68
Enzymes Involved in Homologous Recombination
1. RecA (aside from DNA polymerase, ligase and
single-stranded binding proteins)
central protein involved in homologous recombination promotes the exchange of strands between
homologous DNAs that causes heteroduplexes to form
capable of catalyzing, by itself, the strand exchange
reactions that are central to the formation of Holliday
junctions
action of RecA can be considered in three stages (see
next slide)
found in E. coli
2. RecBCD enzyme (most recombination events in E.coli) complex of 3 proteins (RecB, C and D).
initiates recombination by providing the single-stranded
DNA to which RecA binds by unwinding and nicking
double-stranded DNA .
7/31/2019 Bio 108 Lec 5
65/68
Figure 5.35. Function of the
RecA protein
1. RecA initially binds tosingle-stranded DNA to
form a protein-DNA
filament.
2. The RecA protein that
coats the single-stranded
DNA then binds to a
second, double-stranded
DNA molecule to form a
non-base-paired
complex.
3. Complementary basepairing and strand
exchange follow, forming
a heteroduplex region.
7/31/2019 Bio 108 Lec 5
66/68
Figure 5.36. Initiation of
recombination by RecBCD
1. The E. coliRecBCD
complex binds to the
end of a DNA molecule
and unwinds the DNA
as it travels along the
molecule.2. When it encounters a
specific sequence
(called a chi site*), it
nicks the DNA strand.
3. Continued unwindingthen forms a displaced
single strand to which
RecA can bind.
*specific nucleotide sequence
(GCTGGTGG)
3 R A B d C
7/31/2019 Bio 108 Lec 5
67/68
3. RuvA, B, and C
E. coliproteins become involved in recombination once
a Holliday junction is formed
Figure 5.37. Branch
migration and resolution of
Holliday junctions
1. Two E. coliproteins
(RuvA and RuvB)
together catalyze themovement of the
crossed-strand site in
Holliday junctions
(branch migration).
2. RuvC resolves theHolliday junctions by
cleaving the crossed
strands, which are then
joined by ligase.
RAD51
7/31/2019 Bio 108 Lec 5
68/68
RAD51
-RecA-related protein in yeast
-required for genetic recombination as well as for the repair
of double-strand breaks-able to catalyze strand exchange reactions in vitro
-Proteins related to RAD51 have been identified in complex
eukaryotes, including humans
*In yeasts:Holliday junctions are resolved by a complex of RAD1
and RAD10, with RAD1 cleaving single-stranded DNA at the
crossover junction. (RAD1 and RAD10 are homologs of the
mammalian XPF and ERCC1 DNA repair proteins and also
cleave damaged DNA during nucleotide-excision repair).