DNA Enzymes and Proteins Involved in DNA Replication in
Prokaryotes Slide 2 DNA replication( bacteria ) Initiation
Elongation Termination Daughter DNA partition Slide 3 * the origin
of replication is defined * replication bubble. * Replication fork
* Unidirectional or bidirectional. Origins Slide 4 Slide 5 Figure
13.9 The leading strand is synthesized continuously while the
lagging strand is synthesized discontinuously.
Elongation(semidiscontinuous) Slide 6 termination Slide 7 DNA 1 DNA
2 DNA DNA primase 3 DNA 4 DNA Slide 8 I polA major repair enzyme II
polB minor repair enzyme III polC replicase IV dinB SOS repair V
umuC D SOS repair DNA polymerases in E.coli Slide 9 DNA Polymerase
I DNA polymerase 3 5 exonuclease 5 3 exonuclease Slide 10 Figure
13.8 The catalytic domain of a DNA polymerase has a DNA-binding
cleft created by three subdomains. The active site is in the palm.
Proofreading is provided by a separate active site in an
exonuclease domain. Slide 11 Figure 13.7 Crystal structure of phage
T7 DNA polymerase has a right hand structure. DNA lies across the
palm and is held by the fingers and thumb. Photograph kindly
provided by Charles Richardson and Tom Ellenberger. Slide 12 Figure
13.5 Nick translation replaces part of a pre- existing strand of
duplex DNA with newly synthesized material. DNA Polymerase I Slide
13 Subunit composition of E.coli DNA polymerase III holoenzyme
subunit molecular mass function subassemblies (KDa) 129.9 DNA
polymerase 27.5 3 5 exonuclease core 8.6 stimulates exonuclease
71.1 dimerizes core Pol III binds complex 47.5 binds ATP 38.7 binds
to Pol III 36.9 binds to and complex 16.6 binds to SSB
DNA-dependent 15.2 binds to and ATPase 40.6 sliding clamp Slide 14
E.coli Pol III Beta-subunit Slide 15 Figure 13.18 DNA polymerase
III holoenzyme assembles in stages, generating an enzyme complex
that synthesizes the DNA of both new strands. Slide 16 Fig. 1.
Model of SOS translesion replication by DNA polymerase V. The two
DNA strands are shown as green lines, and the replication-blocking
lesion is represented by the red rectangle. The three major steps
in TLR are pre-initiation (2), in which the RecA nucleoprotein
filaments assembles; initiation (3 and 4), which involves binding
of pol V to the primer- template and loading of the subunit clamp;
and lesion bypass by pol V holoenzyme (5). SSB is suggested to help
in displacing RecA from DNA both at the initiation and lesion
bypass steps. Slide 17 E. coli DNA polymerase IV dinB gene * dinB
DNA * UmuC UmuD Y DNA * E. coli DNA polymerase IV Slide 18 2 DNA
DNA primase Use host RNA polymerase as primase (M13) primosome
primase (dnaG protein) (E.coli) other proteins X174: only primase,
without the other proteins Slide 19 Initiation requires several
enzymatic activities, including helicases, single-strand binding
proteins, and synthesis of the primer. Slide 20 Adenovirus terminal
protein binds to the 5 end of DNA and provides a C-OH end to prime
synthesis of a new DNA strand. Slide 21 A primer terminus is
generated within duplex DNA. Nick translation replaces part of a
pre-existing strand of duplex DNA with newly synthesized material.
DNA Polymerase I Slide 22 DNA Helicase Topoisomerases Slide 23
Helicase 4 helicases * rep helicase * DNA helicase II * DNA
helicase III * dnaB Protein: E.coli DNA DNA Slide 24 Topoisomerases
I(topA gene) act on highly negatively supercoiled DNA stabilize
single-stranded regions Slide 25 Figure 14.16 Bacterial type I
topoisomerases recognize partially unwound segments of DNA and pass
one strand through a break made in the other. Slide 26 II Type II
topoisomerases generally relax both negative and positive
supercoils. The reaction requires ATP Slide 27 Figure 14.17 Type II
topoisomerases can pass a duplex DNA through a double-strand break
in another duplex. Slide 28 IV DNA Slide 29 DNA 1 Original complx:
DnaA DnaB DnaC DnaG HUand SSB Slide 30 The minimal origin is
defined by the distance between the outside members of the 13-mer
and 9-mer repeats Slide 31 Prepriming involves formation of a
complex by sequential association of proteins, leading to the
separation of DNA strands. Slide 32 methylation at the origin Slide
33 A membrane-bound inhibitor binds to hemimethylated DNA at the
origin, and may function by preventing the binding of DnaA. It is
released when the DNA is remethylated. SeqA Slide 34 The complex at
oriC can be detected by electron microscopy. Antibodies of dnaA
Slide 35 protein HU The protein HU is a general DNA-binding protein
in E. coli. Its presence is not absolutely required to initiate
replication in vitro, but it stimulates the reaction. HU has the
capacity to bend DNA, and is likely to be involved in some general
structural capacity. Slide 36 2 (Dna G) (DnaB) Slide 37 2 Tus Slide
38 How do the daughter DNAs become disentangled? Slide 39 DNA DNA
multiple replication * oriC * Slide 40 A simplified model of the
bacterial cell cycle.The model is simplified to ignore multifork
replication. SMC Slide 41 A model of a circular chromosome that is
undergoing multifork replication in a rod-shaped bacterium. Slide
42 Slide 43 1 SeqA Slide 44 2 polymerase, helicase and accociated
proteins PolC-GFP SeqA) H3 pull DNA template duplicated release DNA
outward during replication The extrusion-capture model for
bacterial chromosome partitioning. Slide 45 3 organization
(compaction) (helicase) SMC MukB to organize the chromosome into a
higher order structure by constraining supercoils.(cause)
Partitioning Motor protein (altered) Chromosome
partitioning(consequence) HU; Hbsu ; Slide 46 Terminus- specific
chromosome partitioning events. Tyrosine site-specific recombinases
E.coli CodV, RipX B.subtilis Post-septation partitioning FtsK Slide
47 PBP2 RodA PBP 3 peptidoglycan( ) EnvA Slide 48 Figure 12.27
Failure of cell division generates multinucleated filaments. Slide
49 E. coli generate anucleate cells when chromosome segregation
fails. Cells with chromosomes stain blue; daughter cells lacking
chromosomes have no blue stain. This field shows cells of the mukB
mutant; both normal and abnormal divisions can be seen. Photograph
kindly provided by Sota Hiraga. Minicells: anucleate cells Slide 50
Problem 1. DNA 2. 3. 4. 5. Slide 51 20.
