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Basic Molecular Genetic Mechanisms: (Part 3)

DNA polymerase β is involved in DNA replication and repair. During replication, DNA polymerase adds new nucleotides to build new DNA strands using existing strands as templates. Polymerase α and δ are the main polymerases involved, along with other proteins like primase, helicase, ligase, and topoisomerases. Lagging strand synthesis requires multiple RNA primers and is discontinuous, while leading strand synthesis only requires one primer and is continuous. DNA repair mechanisms like base excision repair, nucleotide excision repair, and double-strand break repair via homologous recombination or end-joining help fix errors from replication or damage.

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57 views35 pages

Basic Molecular Genetic Mechanisms: (Part 3)

DNA polymerase β is involved in DNA replication and repair. During replication, DNA polymerase adds new nucleotides to build new DNA strands using existing strands as templates. Polymerase α and δ are the main polymerases involved, along with other proteins like primase, helicase, ligase, and topoisomerases. Lagging strand synthesis requires multiple RNA primers and is discontinuous, while leading strand synthesis only requires one primer and is continuous. DNA repair mechanisms like base excision repair, nucleotide excision repair, and double-strand break repair via homologous recombination or end-joining help fix errors from replication or damage.

Uploaded by

ofir
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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DNA polymerase

(pol )

Basic
Molecular
Genetic
Mechanisms
(Part 3 )
DNA Replication
and Repair
Molecular Cell Biology
7th ed
Chapter 4

Major Points of DNA Replication


New strand is built 5 3 using an
existing strand as a template (3 5)
DNA polymerase adds new dNTPs
2 different DNA polymerases are involved
(pol and pol )
Only extends 3 end of a duplex (primer)

Numerous other enzymes assist

Primers
Short strand of complementary
nucleotides (DNA or RNA)
synthesized by primase
composed of RNA (8 10 dNTPs long)
extended 5 3 by pol
must be
RNA
excised

Leading versus Lagging Strands


Leading strand (continuous synthesis)
Only one primer required
Replication proceeds toward replication
fork, (DNA Pol )
Lagging strand (discontinuous synthesis)
Requires multiple primers
Replication proceeds away from
replication fork (DNA Pol Pol )

Schematic Diagram of a
Replication Fork

SV40 DNA
Replication Fork

Replication Proteins (1)


helicase (separates parental strands of DNA)
Composed of 6 homologous MCM* proteins
(Large T-antigen in SV40)

Primase (synthesizes short RNA primer)


DNA Polymerases
pol -extends RNA primer on both strands
pol - replaces pol , lagging strand syn.
pol - replaces pol , leading strand syn.
* mini chromosome maintenance complex

Replication Proteins (2)


Replication protein A (RPA) stabilizes ssDNA
heterotrimeric protein, with 2 DNA
binding domains
Ribonuclease H and FEN I (excise
ribonucleotides at 5 end of Okazaki fragments)
Proliferating Cell Nuclear Antigen (PCNA)
(captures daughter dsDNA, prevents PCNARfc-Pol complex from disassociating from
template)

Homotrimeric ring-like protein

Replication Proteins (3)


Replication factor C (RfC) (anchors Pol
or Pol to sliding clamp PCNA)
DNA ligase (re-forms phosphodiester bond
between nucleotides, joins nicks)
Topoisomerases (relieve torsional stress in
dsDNA caused by helicase)

topo I single strand cutter, creates nicks


topo II double strand cutter

DNA Sliding Clamp


(Proliferating cell
nuclear antigen)

PCNA

Eukaryotic Clamp Loader


(Replication factor C)

RfC

SV40
Large T-antigen

Bidirectional
DNA
Replication in
SV40
circular DNA
molecule
1 cut by EcoR1
Center of replication bubble
stationary

Topo I relieves tensional strain

RPA binds ssDNA

Primase synthesis RNA primer

Pol extends primers

PCNA-RfC-Pol complex replaces pol , extends strand

Primase synthesizes new primers for lagging strands

Pol extends primers

PCNA-RfC-Pol complex replaces pol ,


extends strand

Ribonuclease H and FEN 1 excise primers


PCNA-RfC-Pol complex fills in dNTPs
DNA ligase joins fragments

DNA Replication Control


Control of initiation is regulated
Activation of MCM helicase is required
to initiate cellular DNA replication
MCM is regulated by S-phase cyclindependent kinase

Eukaryotes have multiple origins of


replication along each chromosome

DNA Polymerase Coordination


replication fork must coordinate
synthesis of both strands
leading strand synthesis is faster than
lagging strand synthesis
two helicase complexes are connected

discontinuous synthesis of lagging


strand follows Trombone Model

Sources of Errors in DNA


DNA Replication errors
E. coli native error rate 1 x 10-4, observed
error rate in cells 1 x 10-9
DNA polymerase proof reading corrects
many mistakes

Chemical and Radiation damage


deamination can convert 5methylcytosine to thymine

DNA Polymerase Proofreading


5'-3' polymerase site

3'-5' exonuclease site

E. coli: DNA polymerase native error rate 1 x 10-4


Measured mutation rate 1 x 10-9
Animal cells: Pol and Pol both have proofreading

Deamination-Based Point Mutations


Why is cytosine
methylated?

Major Classes of DNA Repair


Enzymes
Single Strand Repair (deals with single
base-pairs)
DNA mismatch repair
Nucleotide excision repair

Double Strand Repair (deals with


larger areas)
Homologous recombination (error free)
End-Joining (error prone)

Base Excision
Repair of G-T Mismatches
1. DNA glycosylase identifies G-T
mismatch, excises N-base (T).
2. APEI endonuclease breaks
phsosphodiester bond freeing 5' end
of baseless deoxyribose.
3. Endonuclease associated with Pol
removes sugar and adds new
nucleotide (C).
4. Ligase re-forms 3' phosphodiester
bond

Human MSH2/MSH6 Based


Mismatch Excision Repair
1. MSH2/MSH6
complex binds to
mispaired segment.
Recognizes template
strand.
2. MLH1 endonuclease
binds daughter strand
with PMS2. DNA
helicase unwinds
DNA. Exonuclease
excises bases.
3. Pol repairs excision.
Ligase rejoins strand

Thymine-Thymine Dimer
Formation

Opening of DNA
double helix

XP-C (xeroderma pigmentosum C)


Mediated DNA Lesion Repair

XP-C (xeroderma pigmentosum C)


Mediated DNA Lesion Repair Steps
1. XP-C/ 23B protein complex identify DNA lesion.
Complex recruits TFIIH transcription factor.
Helicase subunit of TFIIH unwinds DNA
utilizing ATP.
2. XP-G and RPA bind to complex. XP-G and XP-F
cut damaged strand 24-32 bases on either side
of damage.
3. Damage fragment is released and degraded to
mononucleotides.
4. DNA polymerase fills in gap. Ligase seals strand.

Error-Prone Repair
of Double-Strand
Breaks by
End-Joining
1. DNA-PK protein binds ends of
broken strands.
2. DNA synapse formed between
two ends of fragments, and
nucleases remove bases from the
two ends.
3. DsDNA molecules joined by
ligation.
Break repaired, but several
bases at site of break are lost.

Homologous Recombination
Error-free repair of double stranded
breaks in DNA
can repair DNA damage
generate genetic diversity

involves physical exchange of DNA


between DNA molecules
(recombination)

Repair of
Collapsed
Replication Forks
1. Collapse of fork
2. 5' exonuclease erodes
broken end.
3. Strand invasion occurs
4. New bases added causing
branch migration
5. Heteroduplex resolved.
6. Fragments are ligated
7. Rebuilt replication fork
continues replication

Double-Strand DNA Break Repair


by Homologous Recombination (1)
1. Broken ends resected by
5 acting exonuclease
2. RecA- or Rad51mediated strand invasion
occurs
3. 3 end of invading strand
extended by DNA
polymerase until
displaced strand (dark
blue) base-pairs with
other 3 single strand
generated initially (pink)

Double-Strand DNA Break Repair by


Homologous Recombination (2)
4. 3 end is extended
by DNA polymerase. Extended
strand bridges gap in
broken strand.
5. End of newly
synthesized strands
ligated to original
strands.
5. Cleavage of
phosphodiester bonds (arrows) and ligation of alternate
ends restore DNA molecules.

Resolution of A Holliday
Structure
1. regenerates original chromosomes

1. Horizontal cut regenerates original chromosomes


2. Vertical cut produces two recombinant chromosomes

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