DNA Replication, Repair, and

Recombination
Cell division - mitosis
S-phase is tightly regulated by kinases
Mitosis can be divided into six phases
Mitosis can be divided into six phases
 Meselson-Stahl experiment
Proved that DNA replication is semi-conservative
 There are various “problems” that must be
overcome for DNA polymerase to copy DNA

DNA polymerases are unable to melt duplex DNA in order to separate the
two strands that are to be copied

All known DNA polymerases can only elongate a preexisting DNA or

RNA strand (the primer) and are unable to initiate chains

The two strands in the DNA duplex are opposite in chemical polarity, but
all DNA polymerases catalyze nucleotide addition at the 3'-hydroxyl end
of a growing chain, so strands can grow only in the 5' to 3' direction.
Hence, the 3’ – 5’ strand (lagging strand) synthesis cannot be straight
forward

For linear DNA: The ends (telomere regions) of the 3’-5’ strands cannot
be primed. Hence, the replicate strand would be shorter than the parent
strand
Replication fork contains a leading-strand
and lagging- strand
Enzymes involved in replication

1: Helicase unwinds parental DNA strands

2: Single strand regions are bound and stabilized by
multiple copies of the protein RPA (stabilizes a DNA
conformation optimal for processing by DNA pol δ)
3: Leading strand synthesis via an enzymatic
complex:
DNA Pol δ, PCNA, and Rfc
4: Primers for lagging strand synthesis (RNA, DNA)
are synthesized by a complex of DNA pol α and
Primase resulting in a mix RNA-DNA primer
5: The 3’ end of each RNA-primer is then bound by
a PCNA-Rfc-Pol δ complex, which extends the
primer and synthesize most of each Okazaki
fragment (incl proofreading!)
6: Finally the Okazaki fragments are then joined into
a complete lagging strand by the enzyme “Ligase”
Synthesis of the lagging strand

primase

DNA pol α

DNA pol δ

ligase
Replication is bidirectional
 DNA replication begins at specific chromosomal sites
called replication origins

Consensus sequence of the minimal bacterial replication origin

Replication origins, regardless of organism, are

(1) unique DNA segments with multiple short repeats,
(2) recognized by multimeric origin-binding proteins,
(3) usually contain an A-T rich stretch
Type I topoisomerases relax DNA by nicking and
then closing one strand of duplex DNA

The role of topoisomerases in DNA replication

DNA molecules can coil and bend in space,
leading to changes in topology such as formation
of supercoils
Topoisomerases are enzymes that control DNA
topology and perform essential functions at
several different steps in replication
 Replicated circular DNA molecules are
separated by type II topoisomerases

Linear daughter chromatids also are

separated by type II topoisomerases
Telomerase prevents shortening of lagging
strands during eukaryotic DNA replication

Mammalian telomere
Figure
repeat: 12-13
sequence
(TTAGGG)*1000*X
 DNA damage and repair and their role in
carcinogenesis

A DNA sequence can be changed by copying errors introduced by

DNA polymerase during replication and by environmental agents
such as chemical mutagens or radiation

If uncorrected, such changes may interfere with the ability of the

cell to function

Hence, several mechanisms to repair DNA damage have evolved

All carcinogens cause changes in the DNA sequence

--> DNA damage and/or failing repair can lead to cancer
development

Prokaryotic and eukaryotic DNA-repair systems are analogous

General types of DNA damage and causes
 What if Proofreading

- Proofreading is used in genetics to refer to the error-

correcting processes

- First proposed by John Hopfield and Jacques Ninio

- Involved in DNA replication, immune system specificity,

enzyme-substrate recognition - processes that require
enhanced specificity

- The proofreading mechanisms are processes that

consume ATP to enhance specificity of various
biochemical reactions
Prokaryotes Vs Eukaryotes

- In bacteria, all three DNA polymerases (I, II and III) have

the ability to proofread, using 3’ → 5’ exonuclease
activity
- When an incorrect base pair is recognized, DNA
polymerase reverses its direction by one base pair of
DNA and excises the mismatched base
- Following base excision, the polymerase can re-insert
the correct base and replication can continue

- In eukaryotes only the polymerases that deal with the

elongation have proofreading ability (3’→5’
exonuclease activity)
 Proofreading cont….

- Proofreading also occurs in mRNA translation for protein

synthesis
- In this case, one mechanism is release of any incorrect
aminoacyl-tRNA before peptide bond formation
- The extent of proofreading in DNA replication determines the
mutation rate, and is different in different species
- E.g. loss of proofreading due to mutations in the DNA
polymerase epsilon gene results in a hyper-mutated
genotype with >100 mutations per Mbase of DNA in human
colorectal cancers
- The extent of proofreading in other molecular processes can
depend on the effective population size of the species and
the number of genes affected by the same proofreading
mechanism
 Proofreading
by DNA polymerase corrects copying errors

DNA Pol α
Schematic model of the proofreading function
of DNA polymerase
Chemical carcinogens react with DNA directly or after
activation, and the carcinogenic effect of a chemical
correlates with its mutagenicity
Deamination leads to point mutations
Base excision repair of T*G mismatch
Mismatch excision repair in human cells
Formation of thymine-thymine dimers

Sunlight
Space radiation
Nucleotide excision repair in human cells

NB:
Transcription-coupled repair
Nonhomologous end-joining
Double-strand break repair
Double-strand breaks frequently lead to genetic
aberration ... And cancer
Recombinatorial repair of collapsed Replication fork
Double-strand break repair
Homologous recombination (CrossOver)
Double-strand break repair

 Recombination provides a means

by which a genome can change to
generate new combinations of
genes (pro-evolution)

 Homologous recombination allows

for the exchange of blocks of
genes between homologous
chromosomes and thereby is a
mechanism for generating genetic
diversity

 Recombination occurs randomly

between two homologous
sequences and the frequency of
recombination between two sites
is proportional to the distance
between the sites