Chapter 28 discusses DNA replication, repair, and recombination, highlighting the accuracy of DNA synthesis along with proofreading mechanisms that reduce error rates. It details the roles of various enzymes, such as DNA polymerases and topoisomerases, in facilitating efficient DNA replication and repairing damages that can lead to mutations or cancer. The chapter also covers the processes involved in priming and completing DNA synthesis along with mechanisms for correcting errors and repairing DNA damage.

1. Chapter 28:
DNA Replication, Repair, and
Recombination
Copyright © 2007 by W. H. Freeman and Company
Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition

2. Accuracy & Proof Reading
• One (01) bp per 3 X 109
bp
– Through multilayered system
• Accurate DNA Synthesis (1bp per 3 X 103
- 104
bp)
• Proof reading during DNA synthesis (reduces error rate
1bp per 3 X 106
- 107
bp )
• Postreplication mismatch repair (reduces error rate 1bp per
3 X 109
- 1010
bp)
• Even after DNA has been initially replicated, the genome
is still not SAFE
• UV, Chemical species, introducing changes in the DNA
sequence (mutation), or lesion that can block further DNA
replication

3. DNA Repair System
• DNA repair system that detect DNA
damage
• Mutation in the gene the encode
components of DNA repair systems are
key factors in the development of
cancer.
• Devastating type of DNA damage is
double strand breaks in DNA
• With both strand of double helix
broken in a local region, neither strand
is intact to act as template for future
DNA synthesis
• DNA Recombination

4. Basics of DNA Structure
• The two strands of double stranded DNA run in
opposite directions (anti-parallel).
• The purine and pyrimidine bases appended to
the deoxyribose-phosphate polymer strands are
on the inside of the double helix.
• In the Watson-Crick model Adenine (A) base
pairs with Thymine (T) through two H-bonds and
Guanine (G) always base pairs with Cytosine (C)
through three H-bonds.

5. Semiconservative
Replication
of DNA
Based on studies
by Meselson and
Stahl.
Replication always
occurs moving
5'-3'.

6. A and B
DNA
Both are right-
handed helices
B = common,
10.4 bp/turn
base tilt = 5-6o
A = low humidity,
11 bp/turn
base tilt = 11-20o

7. Ribose Conformations
In form A the 3' C is up and in form B the 2' C is up.
Bases are typically anti to the ribose ring so that
base pairing is facilitated.

8. Ribose Attachments Ribose in relationship to
the groves in ds DNA.
The Minor groove contains the pyrimidine O-2 and Purine N-3 of the base pair and
the major groove is on the opposite side of the pair.
Methyl group of thymine lies in the major groove
N-3 of A and G and o-2 of T or C can serve as H bond acceptor and NH2 group
attached to C-2 of G can be a H bond donor
Major groove– N-7 of G or A is a potential acceptor, as are O4 of T and O-6 of G
NH2 gp attached to C-6 of A and C-4 of C can serve as H bond doner

10. Propeller Twist
Study of a short segment of dsDNA (dodecamer)
shows non-coplanarity between some base pairs.

11. DNA, Z Form
A left handed helix with alternating GCGCGC and alternating
syn and anti purines.
Protein domain has been discovered that bind nucleic acids
specifically in the Z form.
This observation strongly suggests that such structures are
present in cells and perform specific function

12. Three Forms of DNA

13. DNA Supercoiling
Relaxed DNA in the B form has 10.4 base pairs
per turn of the helix. The linear structure below
shows the number of turns about the helix axis.

14. Supercoiling Parameters
The Linking number (L) is the number of times
one strand of circular dsDNA passes over the
other. L is constant unless the strands break
and reform.
The Twist (T) is the number of turns that DNA
makes about the duplex axis.
The Writhe (W) is the number of supercoils (turns
about the superhelix axis). A clockwise turn = a
(+) supercoil and ccw = a (-) supercoil.
So: L = T + W

15. Circular
DNA
Closing the linear
DNA seen
previously gives
relaxed, circular
ds DNA with no
supercoils so:
L = T + W
25 = 25 + 0

16. Changing L
Open the circular DNA and unravel two turns.
This decreases L by two.

17. Circular
DNA
Closing the DNA
makes gives
relaxed, circular
ds DNA with no
supercoils, now:
L = T + W
23 = 23 + 0

18. Introducing Supercoils
Making two right-
handed coils in
the previous
helix without
breaking the
strands is equal
to a W of –2 and
T changes:
23 = 25 - 2

19. Electron Micrograph
E.Coli has about 5 supercoils per 1000 bp.

20. Topoisomerase Prepare the Double helix for Unwinding
• Most naturally occuring DNA molecules are negatively supercoiled.
• Negatively supercoiling prepares DNA for processes requiring
separation of the DNA strands, like replication
• Positive super coiling condenses DNA as effectively, but it makes
strand separation more difficult
• Specific enzymes called topoisomerases that introduce or eliminate
supercoils
• Type 1 topoisomerases catalyze the relaxation of supercoiled DNA, a
thermodynamically favorable process
• Type 2 topoisomerases utilize free enerty from ATP hydrolysis to add
negetive supercoils to DNA

21. Topoisomerases alter the Lk number
• Three step process
1. The cleavage of one or both strands of DNA
2. The passage of a segment of DNA through this break
3. The resealing of the DNA break
• Type 1 topoisomerases cleave just one strand of DNA,
whereas, type II enzyme cleave both strands

22. Topoisomerase I
• Human type 1 comprises
four domains having central
cavity (20Ao
)
• This cavity also includes a
tyrosine residue (Tyr 723),
which acts as a nuclephile to
cleave the DNA backbone in
the course of catalysis

23. Tyr of Topoisomerase I
Tyr of Topo I cleaves one strand of DNA to permit
unwinding and a change the linking number (L).

24. Topoisomerase II
Topo II breaks
both strands of
DNA and
requires ATP.
Topo I breaks
only one strand.

25. • The bacterial topoisomerases II (often called DNA gyrase)
is the target of several antibiotics that inhibit the
prokaryotic enzyme much more than the eukaryotic one
• Novobiocin blocks the binding of ATP to gyrase.
• Nalidixic acid and ciprofloxacin, in contrast interfere with
the breakage and rejoining of DNA chains.
• These two gyrase inhibitors are widely used to treat UTI
including those due to Bacillus antracis (anthrax).
• Campthethecin, an antitumor agent, inhibits human
topoisomerase I by stablizing the form of enzyme
covalentaly

26. Complication of DNA Replication
• Two strands run in opposite direction
– Replication 5` to 3` direction, so must have special mechanism to
accommodate the oppositely directed strand
• The two strands of double helix interact with one another
in such a way that the edges of the bases on which the
newly synthesized DNA is to be assembled and occupied.
– Thus two strand must be separted
• The two strands of double helix wrap around each other
– Thus strand seperation also entails the unwinding of the double
helix. This unwinding creates supercoils that must themselves be
resolved as replition continues.

27. DNA Polymerases Requires a Template and a
Primer
• DNA polymerases are template directed enzymes.
• Primer: The initial segment of a polymer that is to be
extended on which elongation depends.
• Template: A sequence of DNA or RNA that directs the
sysnthesis of a complementary sequence

28. DNA Polymerase I
DNA Pol I was the first polymerase isolated. This
was obtained from E.coli by Arthur Kornberg
and is known as the Kornberg enzyme.
This enzyme has a 5'-3' exonuclease activity that
cleaves RNA primers, a 5'-3' polymerase activity
that makes DNA and a 3'-5' exonuclease activity
that repairs DNA.
The Klenow fragment is the large portion after
cleaving off the 5'-3' exonuclease and has been
used as a polymerase in lab work.

29. DNA
Polymerase
This is the
Klenow fragment
of the E.coli
enzyme.

30. DNA
Polymerase
Activity
Note the two Mg++
ions binding
substrate in this
mechanism.

31. Molecular Shape vs H-Bond
Both of these direct thymine into a DNA strand
even though the one cannot form H-bonds.

32. Conformational Change
When the correct dNTP binds a change occurs
resulting in a tight fit for the proper dNTP shape.

33. RNA primer synthesized by Primase
enables DNA synthesis to Begin
• DNA Polymerase cannot initiate DNA synthesis without a
primer.
• RNA primes the synthesis of DNA .
• RNA polymerase called primase synthesizes a short
stretch of RNA (5nt).
• Primase, like other RNA polymerases can initiate synthesis
without a primer.
• After DNA synthesis has been initiated, the short stretch of
RNA is removed by hydrolysis and replaced by DNA

34. The site of DNA synthesis is called replication fork
DNA ligase joins ends of DNA in duplex regions

35. DNA
Helicase
A helicase unwinds
DNA as part of the
primosome.
Bacterial helicases
called PcrA (4
domains)
A primosome is a protein complex responsible for creating RNA primers
on single stranded DNA during DNA replication

36. DNA Helicase
Helicase unwinding of dsDNA requires ATP.

37. E.coli Replication
Three Steps:
1. Initiation: Ori site in E.coli = Ori C
This is a 245 bp highly conserved seq.
2. Polymerization: chain elongation in the 5’-
3’ direction.
3. Termination: “ter region” is ~350 kbp
sequence that is 180o
from Ori C.

38. E.coli OriC Site
The three AT rich sequences on 5' end of OriC are
weak, only two H-bonds per bp. To the right of this
are five-9 bp sequences that bind DnaA (initiation
factor). These five sites have opposing sequences.

39. Priming Events
DnaA binds to the 9 bp sequences along with ATP
and causes opening in the AT rich region. HU a
histone-like protein prevents DnaA from binding
at sites other than OriC.
When the loop is open DnaB, a helicase, binds at
each fork as a complex (DnaB6•DnaC6•ATP6).
DnaT assists and this is the pre-priming
complex.
DnaB continues to unwind increasing the bubble
size displacing DnaA as it moves. SSB binds to
ssDNA to prevent annealing. Topo II binds ahead
of the fork to relieve stress cause by opening.

40. Possible DnaA Binding
Proposals
suggest that
up to 20 or
more DnaA
are bound
and that a
nucleosome
type
structure is
formed.

41. Priming Events
PriA, PriB and PriC enter the bubble along with
DnaG (DNA primase). This completes the
primosome which makes RNA primers. Topo II
and SSB are not part of the primosome. DNA
primase does not need a primer to begin
synthesis of RNA primers.
The primers (10-30 bp) begin at the center base of
any GTT sequence and start a primer about every
1000 bp. Only one primer is needed on the
leading strand. After this is made, the
primosome moves to the lagging strand.

42. Replication Events
Two Pol III holoenzymes (DnaE) enter with a few
other proteins to complete the replisome. Pol III
is an asymmetric dimer that synthesizes DNA
from both template strands simultaneously.
Pol III needs a primer to begin synthesis. The
leading strand is synthesized continuously and
the lagging strand discontinously. Both are
synthesized in the 5'-3' direction.
Bidirectional synthesis occurs at both replication
forks.

43. DNA Pol III, the Replicase
Pol III forms a
sliding clamp
around ds
DNA.
This enzyme is
the workhorse
of replication
and is very
processive.

44. Priming DNA Synthesis
A primer is require to start DNA synthesis.

45. Leading and Lagging Strand
Synthesis
Okazaki fragments are made on the lagging strand.

46. Layout of Participants
General
arrangement
of replication
participants.

48. Synthesis on both Strands
POL III forms
a sliding
clamp around
ds DNA. This
enzyme is
very
processive.

49. Lagging Strand Synthesis
The lagging strand loops to enable both Pol III
core units to move in the direction of the
replication fork.
The lagging strand begins replication at a primer
and proceeds until it runs into another Okazaki
fragment. At this point the core unit dissociates,
the chain shifts to position another primer, the
core rebinds and makes another Okazaki piece.
The lagging strand will always be a little behind
the leading strand.

50. Joining Okazaki Fragments
Joining these fragments requires DNA ligase
after nick translation has occured.

51. Steps for Joining Okazaki
Fragments
DNA ligase seals the nicks using NAD+
for energy
after Pol I has removed RNA. Some organisms
use ATP to adenylate the ligase.
E-lys + NAD+
--> AMP-NHlys-E + NMP
5'-p-DNA + AMP-NHlys-E --> AMP-5'P-DNA + E-lys
AMP-5'P-DNA + 3'OH-DNA --> DNA(sealed) + AMP

52. E.coli Termination
The E.coli ''ter region'' is a ~350 kbp sequence
180o
from Ori C. It contains seven sequences,
TerA to TerG, which are binding sites for ''tus'',
terminator utilization substance. The Ter
sequences are ~ 20 bp long and contain the
conserved sequence 5'-GTGTGTTGT-3'. When
tus is bound replication stops by blocking the
helicase.
G F B C A D E
5' ------------------------------------™------™------
™------- 3'
Ter region (~4.5 min on clock face)

53. E.coli Termination
The clockwise replication is stopped at ter B,C,F or
G and counterclockwise replication at ter A,D or
E. The process is complete when synthesis
from the opposite direction reaches the stopped
strand.
At this point, the two new DNAs are intertwined
and Topo II mediates unraveling these by
cleavage and reassembly.

55. Replication Comparison
Procaryotic Eucaryotic
speed 1000 b/sec 50 b/sec
Okazaki 1000 b 100-200 b
primer ~30 b ~3-5 b
ori sites single multiple
Pol nuclease no nuclease

56. Telomeres
A sequence at the ends of linear eucaryotic
chromosomes that helps stabilize the chromosome.
In humans this is a repeat of AGGGTT and is added
to the ends of the chromosome by the enzyme
telomerase. Telomerase, a reverse transcriptase,
contains an RNA component that codes for the
telomere.

57. Telomere
Synthesis
Telomerase
has an RNA
template.

58. Telomere
Synthesis

59. Mutations
Fidelity is good ~ 1:109
. Both Pol I & III have
proofreading/correction capability.
Mutation: permanent alteration, damage that
escapes repair
Substitutions (silent): Transversion replaces pur
with pyr or pyr with pur. Transition replaces pur
with pur or pyr with pyr.
Frameshift (lethal): Addition adds an extra base,
elongates DNA. Deletion removes a base and
shortens DNA. These change every triplet.

60. DNA Damage
Examples of sources of damage.
Deamination: nitrous acid.
Methylation: N-Me-N-nitrosourea or
Dimethylnitrosamine (Me2N-N=O)
Intercalation: Polynuclear aromatic hydrocarbons
Uv damage: Photodimerization of T, loss of base
(AP formation), phosphodiester cleavage.
Strand breakage: uv or x-rays

61. Oxidation
May lead to errors in H-bonding association and
base pair mismatch.

62. Deamination
NaNO2 + HCl generates nitrous acid, which
converts a primary aromatic amine to a carbonyl.

63. Uv Dimerization
This is a photolytic 2 + 2 cycloaddition reaction.

64. Correction by Pol III
Pol III has exonuclease activity that allows
correction of base pairing errors. Pol I also has
proofreading capability. DNA is the only
biopolymer repaired.

65. Direct Repair
Direct Repair:
Photolyase, a photoreactivating enzyme that
reverses a uv induced thymine dimer (needs vis).
Insertase, is an enzyme that can replace a
specific base at AP site.
O6
-methylguanine methyltransferase is a
suicide enzyme (TON = 1) that transfers methyl
from O6
-methylguanine to a Cys on the enzyme
and as a result loses activity.

66. Excision Repair
Excision Repair: (1. base and 2. nucleotide)
Base: Deamination or methylation may
modify a base. A glycosylase (AlkA) recognizes
and cleaves the modified base to produce an AP
site. AP endonuclease cleaves the strand, Pol I
fills and DNA ligase seals the gap. Note: thermal
effects spontaneously produce AP sites ~ 5 x 103
per day.
Nucleotide or general: Exinuclease
(excision repair endonuclease) cleaves both sides
of the damaged site making a ssDNA gap, Pol I
fills and DNA ligase seals the gap.

67. Glycosylase
AlkA in E.coli.
About 20 of
these enzymes
are known.
There is one
specific for
uracil.

68. Base Excision
Repair
Uracil Glycosylase:
Deamination of
cytosine gives uracil.
This error is corrected
by base excision
repair. Leaving uracil
in place would
produce a C-G to U-T
transition.

69. Nucleotide
Excision
Repair

70. Mismatch Repair
Mismatch Repair:
Focus is primarily on correcting non-
Watson-Crick base pairs called Hoogsteen base
pairs. C-C is least responsive to repair and T-G
is the easiest. The newly made strand is
corrected to match the template. This involves
three proteins: MutS, Mut L and Mut H. MutS
binds to the mismatch, then Mut H binds to 6-MeA
in GATC of the parent near the mismatch and
cleaves. MutL links MutS and MutH. Exonuclease
I removes the mismatch segment 3’-5’. Pol III
comes in and fills in from the template and DNA
ligase seals the gap.

71. Mismatch
Repair

72. Diseases Associated with defective Repair system
• Defects in DNA-repair systems are expected to increase
the overall frequency of mutations and, hence, the
likelihood of a cancer-causing mutation
• There are many genetic disease associated with defective
DNA repair
Xeroderma pigmentosum
• Xeroderma pigmentosum, a rare human skin disease, is
genetically transmitted as an autosomal recessive trait.
• The skin in an affected homozygote is extremely
sensitive to sunlight or ultraviolet light. The skin
becomes dry, and there is a marked atrophy of the
dermis.
• Keratoses appear, the eyelids become scarred, and the
cornea ulcerates. Skin cancer usually develops at several
sites.

73. • Many patients die before age 30 from metastases of these
malignant skin tumors
• In normal cell, half the pyrimidine dimers produced by
ultraviolet radiation are excised in less than 24 hours.
• In contrast, almost no dimers are excised in this time interval
in cells derived from patients with xeroderma pigmentosum
Hereditary nonpolyposis colorectal cancer (HNPCC, or Lynch
syndrome)
• HNPCC results from defective DNA mismatch repair.
HNPCC is not rare as many as 1 in 200 people will develop
this form of cancer
• HNPCC results due to defects in mismatch repair system,
which leads to the accumulation of mutations throughout the
genome. In time, genes important in controlling cell
proliferation become altered, resulting in the onset of cancer.

74. Huntington Disease
Autosomal dominant neurological disorder with a
variable age of onset.
The mutated gene in this disease expresses a
protein in the brain call huntington, which
contains a stretch of consecutive glutamine
residue.
These glutamine residue are encoded by a
tandem array of CAG sequences within the gene.
Unaffected person: 6-31 repeats
Affected person: 36-82 repeats

75. The SOS Response
 Agents that damage DNA, such as UV radiatin, alkylating
agents, cross-linking agents and replication fork collapse,
induces a complex system of cellular changes in E. coli known
as the SOS response.
 E. coli so treated cease dividing and increase their capacity to
repair damaged DNA.
 When E. coli cells are exposed to DNA damaging agents or
agents which can inhibit the replication, a protein RecA after
binding to the ssDNA (which is an indication of DNA demage),
specifically mediates the proteolytic self-cleavage of LexA
protein

76. The SOS Response
 LexA protein represses the expression of SOS
response proteins
 Its binds on SOS box a 20 nt long sequence present
upstream of all proteins participates in SOS
response
 This sequence functions as operator of SOS
response proteins and LexA protein it self.
 During normal growth, LexA largely represses the
expression of the SOS genes and it self too by
binding on SOS box to inhibit the RNA polymerase
to start transcription.

77. The SOS Response
 When DNA damage is has sufficient to produce
postreplication gaps, the damaged ssDNA binds to RecA
to stimulate the cleavage of LexA and release of SOS
boxes from repression.
 The SOS response proteins express, including LexA,
which is continually cleaved by RecA.
 When the lesions have been eliminated, RecA ceases
simulating the self-cleavage of LexA, which now starts
to repress the expression of SOS proteins.
 The repair is called Translesion synthesis (TLS).

78. The SOS Response
 The repair is filled by error-rone DNA Pol IV or V.
 The DNA Pol V is also called Pol V mutasome tends to
incorporates G about half as often as A opposite thymine
dimmers and AP sites, with pyrimidines installed
infrequently.
 In TLS some time deletion and insertions also take place
resulting in Frameshift mutations.
 After synthesizing about 7 nt, the DNA Pol V
mutasome is replaced by Pol III holoenzyme, which
replicate DNA about 1000 fold more fidelity.

79. The SOS Response
• Similarly, Pol II with high fidelity also
participate in TLS and express before the
DNA Pol V, the role of DNA Pol II is to
mediate the error-free TLS, and only if this
process fails, it is replaced by DNA Pol V
to carry out error-prone TLS.
• The remaining nick after filling the gap is
filled by DNA ligase.

80. The SOS Response
 SOS repair is an error-prone and hence mutagenic
process.
 It is therefore a last resort that is only initiated about 50
minutes after SOS induction, if the DNA has not already
been repaired by other mechanisms.
 In E. coli, most mutations arise from the action of the
SOS repair system
 Under conditions of environmental stress, the SOS
system functions to increase the rate of mutation so as to
increase the rate at which the E. coli adapt to the new
conditions.

81. SOS Response
SOS Response:
Repair in this instance almost always results
in inaccurate or “error prone” production of a
daughter strand due to polymerization through an
unreadable template. As a result, a mutant is
produced. The error(s) produced in the daughter
strand may or may not be lethal, however this is
the only path available. The alternative is cell
death caused by incomplete replication.

82. SOS Response
SOS Response:
A protein, LexA, normally binds to the SOS
box to prevent expression. Major DNA damage
results in RecA protein binding to the ssDNA and
this in turn promotes autolysis of LexA. The SOS
box is now unprotected and is expressed. The
resulting SOS proteins are capable of repairing
large segments of DNA, even filling in gaps where
bases are missing.

83. Recombination
This is sometimes called post-replication repair.

86. End of Chapter 28
Copyright © 2007 by W. H. Freeman and Company
Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition