BIOLOGI MOLEKULER3. REPLIKASI DNA dan OKAZAKI FRAGMENT

drh. Fajar Shodiq Permata, M.Biotech

3. REPLIKASI DNA dan OKAZAKI FRAGMENT

SUBPOKOK BAHASAN• PROSES REPLIKASI DNA• OKAZAKI FRAGMENT

TUJUAN PEMBELAJARAN1. Mahasiswa mampu memahami proses

replikasi DNA dan Okazaki fragment

REFERENSI

• Alberts, B, et al., 2008, Molecular Biology of The Cell, 5th ed., Garland Science Taylor and Francis Group: UK

• Cooper, G.M., and Hausman, R.E., 2007, The Cell: A Molecular Approach, 4th ed., ASM Press: Washington D.C.

• Alberts, B., et al., 2013, Essential Cell Biology, Garland Science: UK

REPLIKASI DNA

• In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNAmolecule.

• DNA is made up of a double helix of two complementary strands.

https://en.wikipedia.org/wiki/Molecular_biologyhttps://en.wikipedia.org/wiki/DNAhttps://en.wikipedia.org/wiki/Nucleic_acid_double_helix

REPLIKASI DNA

• During replication, these strands are separated.

• Each strand of the original DNA molecule then serves as a template for the production of its counterpart, a process referred to as semiconservative replication.

• Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.

https://en.wikipedia.org/wiki/Semiconservative_replicationhttps://en.wikipedia.org/wiki/Proofreading_(Biology)

DNA REPLICATION

REPLIKASI DNA

• In a cell, DNA replication begins at specific locations, or origins of replication, in the genome.

• Unwinding of DNA at the origin and synthesis of new strands results in replication forks growing bi-directionally from the origin.

• Most prominently, DNA polymerase synthesizes the new strands by adding nucleotides that complement each (template) strand.

• DNA replication occurs during the S-stage of interphase.

https://en.wikipedia.org/wiki/Cell_(biology)https://en.wikipedia.org/wiki/Origin_of_replicationhttps://en.wikipedia.org/wiki/Genomehttps://en.wikipedia.org/wiki/Replication_forkhttps://en.wikipedia.org/wiki/DNA_polymerasehttps://en.wikipedia.org/wiki/Nucleotideshttps://en.wikipedia.org/wiki/Interphase

CELL CYCLE

DNA REPLICATION

DNA POLYMERASE

• In general, DNA polymerases are highly accurate, with an intrinsic error rate of less than one mistake for every 107 nucleotides added.

• In addition, some DNA polymerases also have proofreading ability; they can remove nucleotides from the end of a growing strand in order to correct mismatched bases.

DNA POLYMERASE

• Finally, post-replication mismatch repair mechanisms monitor the DNA for errors, being capable of distinguishing mismatches in the newly synthesized DNA strand from the original strand sequence.

• Together, these three discrimination steps enable replication fidelity of less than one mistake for every 109 nucleotides added.

PROOFREADING DNA

by DNA POLYMERASE

TAHAPAN REPLIKASI DNA

Initiation

Elongasi

Terminasi

INITIATION STAGE

• For a cell to divide, it must first replicate its DNA.

• This process is initiated at particular points in the DNA, known as "origins", which are targeted by initiator proteins.

• Sequences used by initiator proteins tend to be "AT-rich" (rich in adenine and thymine bases), because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair) and thus are easier to strand separate.

https://en.wikipedia.org/wiki/Cell_divisionhttps://en.wikipedia.org/wiki/Origin_of_replicationhttps://en.wikipedia.org/wiki/Initiator_protein

INITIATION STAGE

• Once the origin has been located, these initiators recruit other proteins and form the pre-replication complex, which unzips the double-stranded DNA.

https://en.wikipedia.org/wiki/Pre-replication_complex

INIATION STAGE

• Topoisomerase isolated the replication site

• Spesific Protein binding with Helicase unzips the DNA

• Forming replication fork first by helicase, which break the hydrogen bonds holding the two DNA strands together

• RNA primer (A-T rich) as initiator and attach with DNA because of DNA primase

PRE-REPLICATION COMPLEX

• Topoisomerase-Binding proteins-Helicase-DNA primase

REPLICATION FORK

• Replication fork was formed by Helicase

• It is contained :

a. Leading strand : continous

b. Lagging strand : segmented Okazaki fragment

REPLICATION FORK

• Scheme of the replication fork.

a: template, b: leading strand, c: lagging strand, d: replication fork, e: primer, f: Okazaki fragments

https://en.wikipedia.org/wiki/Okazaki_fragments

ELONGATION STAGE

• Elongation by DNA polymerase

• DNA polymerase has 5'-3' activity.

• note: the DNA template is read in 3' to 5' direction whereas a new strand is synthesized in the 5' to 3' direction

3’-5’ 5’-3’

REPLICATION FORK

• Replication fork was formed by Helicase

• It is contained :

a. Leading strand : continous

b. Lagging strand : fragmented

LEADING STRAND

• The leading strand is the strand of nascent DNA which is being synthesized in the same direction as the growing replication fork.

• A polymerase "reads" the leading strand template and adds complementary nucleotides to the nascent leading strand on a continuous basis.

https://en.wikipedia.org/wiki/Nucleotides

LAGGING STRAND

• The lagging strand is the strand of nascent DNA whose direction of synthesis is opposite to the direction of the growing replication fork.

• Because of its orientation, replication of the lagging strand is more complicated as compared to that of the leading strand.

LAGGING STRAND

• As a consequence, the DNA polymerase on this strand is seen to "lag behind" the other strand.

OKAZAKI FRAGMENT

• A DNA polymerase extends the primed segments, forming Okazaki fragments.

• The RNA primers are then removed and replaced with DNA, and the fragments of DNA are joined together by DNA ligase.

https://en.wikipedia.org/wiki/Okazaki_fragmenthttps://en.wikipedia.org/wiki/DNA_ligase

OKAZAKI FRAGMENT

• The lagging strand is synthesized in short, separated segments (Okazaki fragments).

• On the lagging strand template, a primase"reads" the template DNA and initiates synthesis of a short complementary RNAprimer.

https://en.wikipedia.org/wiki/Primasehttps://en.wikipedia.org/wiki/RNA

3’-5’ 5’-3’

OKAZAKI FRAGMENT

TERMINATION STAGE

• Termination requires that the progress of the DNA replication fork must stop or be blocked.

• Termination at a specific locus, when it occurs, involves the interaction between two components:

(1) a termination site sequence in the DNA, and (2) a protein which binds to this sequence to physically stop DNA replication.

• this is named the DNA replication terminus site-binding protein, or Ter protein.

https://en.wikipedia.org/wiki/Ter_protein

DNA REPLICATION PROTEIN

Enzyme Function in DNA replication

DNA HelicaseAlso known as helix destabilizing enzyme. Helicaseseparates the two strands of DNA at the Replication Forkbehind the topoisomerase.

DNA Polymerase

The enzyme responsible for catalyzing the addition of nucleotide substrates to DNA in the 5' to 3' direction during DNA replication. Also performs proof-reading and error correction. There exist many different types of DNA Polymerase, (such as the DNA Polymerase III )each of which perform different functions in different types of cells.

DNA clampA protein which prevents elongating DNA polymerases from dissociating from the DNA parent strand.

Single-Strand Binding (SSB) Proteins

Bind to ssDNA and prevent the DNA double helix from re-annealing after DNA helicase unwinds it, thus maintaining the strand separation, and facilitating the synthesis of the nascent strand.

https://en.wikipedia.org/wiki/DNA_Helicasehttps://en.wikipedia.org/wiki/Replication_Forkhttps://en.wikipedia.org/wiki/DNA_Polymerasehttps://en.wikipedia.org/wiki/DNA_clamphttps://en.wikipedia.org/wiki/Single-strand_binding_protein

DNA REPLICATION PROTEIN

Enzyme Function in DNA replication

Topoisomerase Relaxes the DNA from its super-coiled nature.

DNA GyraseRelieves strain of unwinding by DNA helicase; this is a specific type of topoisomerase

DNA LigaseRe-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging strand.

PrimaseProvides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the new DNA strand.

Telomerase

Lengthens telomeric DNA by adding repetitive nucleotide sequences to the ends of eukaryotic chromosomes. This allows germ cells and stem cells to avoid the Hayflick limit on cell division.[23]

https://en.wikipedia.org/wiki/Topoisomerasehttps://en.wikipedia.org/wiki/DNA_Gyrasehttps://en.wikipedia.org/wiki/DNA_Ligasehttps://en.wikipedia.org/wiki/Okazaki_Fragmentshttps://en.wikipedia.org/wiki/Primasehttps://en.wikipedia.org/wiki/Telomerasehttps://en.wikipedia.org/wiki/Eukaryotic_chromosome_fine_structurehttps://en.wikipedia.org/wiki/DNA_replication

REPLIKASI DNA IN VITRO

• DNA replication can also be performed in vitro(artificially, outside a cell).

• DNA polymerases isolated from cells and artificial DNA primers can be used to initiate DNA synthesis at known sequences in a template DNA molecule.

• The polymerase chain reaction (PCR), a common laboratory technique, cyclically applies such artificial synthesis to amplify a specific target DNA fragment from a pool of DNA.

https://en.wikipedia.org/wiki/In_vitrohttps://en.wikipedia.org/wiki/Polymerase_chain_reaction

POLYMERASE CHAIN REACTION (PCR)

TERIMA KASIH

SOURCE : https://en.wikipedia.org/wiki/DNA_replicationhttps://en.wikipedia.org/wiki/Okazaki_fragments

https://en.wikipedia.org/wiki/DNA_replicationhttps://en.wikipedia.org/wiki/Okazaki_fragments