DNA repair

Introduction
• Injury to DNA is minimized by systems that recognize
• Correct the damage
• The repair systems are as complex as the replication apparatus itself
• Indicates their importance for the survival of the cell
• When a repair system reverses a change to DNA
• There is no consequence
 Introduction
• Repair systems often can recognize a range of distortions in DNA
as signals for action
• A cell is likely to have several systems able to deal with DNA
damage
• The importance of DNA repair in eukaryotes is indicated by the
identification of > 130 repair genes in the human genome
 Systems divided into several general types

Some enzymes directly reverse specific sorts of

damage to DNA

There are pathways which function by removing and

replacing material:
1. base excision repair
2. nucleotide excision repair
3. mismatch repair
 Systems divided into several general types
There are systems that function by using recombination to retrieve an
undamaged copy
Then used to replace a damaged duplex sequence.

The non-homologous end-joining pathway rejoins

broken double-stranded ends

Several different DNA polymerases can

resynthesize stretches of replacement DNA
 Systems divided into several general types
• Direct repair is rare
• involves the reversal or simple removal of the damage
• Example: Photo reactivation of pyrimidine dimers
• inappropriate covalent bonds b/w adjacent bases are reversed by a light
dependent enzyme
• This system is widespread in nature, occurring in all but placental
mammals, and appears to be especially important in plants
• In E. coli it depends on the product of a single gene (phr) codes for
enzyme photolyase
 Excision repair
Removal of incorrect or damaged sequences followed
by repair synthesis
Excision repair pathways are initiated by recognition
enzymes that see an actual damaged base or a
change in the spatial path of DNA
Some excision repair pathways recognize general
damage to DNA; others act upon specific types of base
damage
There are usually multiple excision repair systems in a
single cell type
 Mismatch repair (MMR)
• Mismatches between the strands of DNA are one of the major targets
for excision repair systems
• MMR is accomplished by scrutinizing DNA for apposed bases that do
not pair properly
• This system also recognizes insertion/deletion loops in which
sequences present in one strand that are absent in the
complementary strand are looped out.
• MM and insertion/deletion loops that arise during replication are
corrected by distinguishing between the “new” and “old” strands
• preferentially correcting the sequence of the newly synthesized
strand
• Other systems deal with MM generated by base conversions
(deamination)
 Two major excision repair pathways
• In addition to mismatch repair
• Base excision repair (BER) systems
• Directly remove the damaged base and replace it in DNA
• A good example is DNA uracil glycosylase,
• Removes uracils that are mispaired with guanines
 Two major excision repair pathways
• Nucleotide excision repair (NER) systems
• Excise a sequence that includes the damaged base(s)
• A new stretch of DNA is then synthesized to replace the excised
material
 Damage that trigger repair systems
• Divided into two general classes
• Single-base changes
• Affect the sequence of DNA but do not grossly distort its overall
structure
• They do not affect transcription or replication, when the strands of
the DNA duplex are separated
• These changes exert their damaging effects on future generations
through the consequences of the change in DNA sequence
• The reason for this type of effect is the conversion of one base into
another that is not properly paired with the partner base
• Single-base changes may happen as the result of mutation of a base
in situ or by replication errors
 Single-base changes (Deamination)
• Deamination of cytosine to uracil (spontaneously or by chemical
mutagen) creates a mismatched U-G pair
 Single-base changes (Mismatched pair)
• A replication error might insert adenine instead of cytosine to create an
A-G pair
 Single-base changes
• Similar consequences could result from covalent addition of a small
group to a base that modifies its ability to base pair
• These changes may result in very minor structural distortion (as in
the case of a U-G pair) or quite significant change (as in the case of
an A-G pair)
• But the common feature is that the mismatch persists only until the
next replication
• Thus, only limited time is available to repair the damage before it is
made permanent by replication
 Structural distortions
• Provide a physical impediment to replication or transcription.
• Introduction of covalent links between bases on one strand of DNA or
between bases on opposite strands inhibits replication and transcription
• The example of UV irradiation,
which introduces covalent
bonds b/w two adjacent Py
bases
• Results in an intrastrand Py
dimer, T-T dimers (most
common), C-C (least common)
 Structural distortions
• Similar consequences can result from the addition of a bulky adduct
(a methyl group) to a base
• That distorts the structure of the double helix
 Structural distortions (Depurination)
• A single-strand nick or the removal of a base prevents a strand from
serving as a proper template for synthesis of RNA or DNA
• The common feature: the damaged adduct remains in the DNA and
continues to cause structural problems and/or induce mutations
• Until it is removed
 Excision Repair Systems in E. Coli

• Incision step, the damaged structure is

recognized by an endonuclease
• That cleaves the DNA strand on both sides of
the damage
 Excision Repair Systems in E. Coli
Excision step, a 5'-3' exonuclease removes a stretch
of the damaged strand
Alternatively, a helicase can displace the damaged
strand, which is subsequently degraded
Synthesis step, SS region serves as a template for a
DNAP to synthesize a replacement for the excised
sequence
Synthesis of the new strand can be associated with
removal of the old strand, in one coordinated action
Finally, DNA ligase covalently links the 3' end of the
new DNA strand to the original DNA
 E. coli uvr system of excision repair
• Accounts for 99% excision repair, average length of replaced DNA is -12
ntds
• The remaining 1% involves the replacement of stretches of DNA mostly -
1500 ntds long, but extending as much as >9000 ntds

• Includes 3 (uvrA, -B, and -C),

• Code for the components of a repair
endonuclease
• UvrAB dimer recognizes pyrimidine
dimers and other bulky lesions
• UvrA dissociates using ATP
• UvrC joins UvrB
 E. coli uvr system of excision repair
• The UvrBC complex makes an
incision on each side using ATP:
• One that is seven ntds from the 5'
side of the damaged site
• Another that is 3-4 ntds away
from the 3' side
• UvrD helicase- helps to unwind the
DNA to allow release of the single
strand between the two cuts
• DNAP I- excises the damaged strand
and repair
 Eukaryotic Nucleotide Excision Repair
Pathways
• The general principle similar to that of bacteria
• Bulky lesions, recognized and repaired by a nucleotide excision repair
system
• The critical role of mammalian nucleotide excision repair is seen in
certain human hereditary disorders
• The best investigated of these is Xeroderma Pigmentosum (XP)
• A recessive disease resulting in hypersensitivity to sunlight (UV) light
• The deficiency results in skin disorders and cancer predisposition
 Eukaryotic Nucleotide Excision Repair
Pathways
• The disease is caused by a deficiency in nucleotide excision repair
• XP patients cannot excise pyrimidine dimers and other bulky adducts
• Mutations occur in one of eight genes called XPA to XPG
• All encode proteins involved in various stages of nucleotide excision
repair
Two pathways of nucleotide excision repair
in eukaryotes
• Global genome repair (GG -NER)
• Transcription-coupled repair (TC-NER)
• The major difference between the two pathways is how the damage is
initially recognized.
 Global genome repair (GG -NER)
• XPC protein detects the damage and initiates the repair pathway
• XPC can recognize damage anywhere in the genome
• XPC component of a lesion-sensing complex, includes the proteins HR23B
and centrin2
• Detects distortions that are not repaired by NER
• Suggesting other proteins are required to verify the damage bound by XPC
• XPC recognizes many types of lesions
• UV -induced cyclobutane pyrimidine dimers (CPDs) not well recognized by
XPC.
• DNA damage-binding (DDB) complex assists in recruiting XPC to this type
of damage
 Transcription-coupled repair (TC-NER)
• Responsible for repairing lesions that occur in the transcribed strand
of active genes
• Damage is recognized by RNA polymerase II itself
• Stalls when it encounters a bulky lesion
• Repair function may require modification or degradation of RNAP
• The large subunit of RNA polymerase is degraded when the enzyme
stalls at sites of UV damage
Global genome repair Transcription-coupled repair
Mismatch errors occur during replication in
E. coli
• Possible to distinguish the original strand of DNA
• Immediately after replication of methylated DNA, only the original
parental strand carries methyl groups
• In the period during which the newly synthesized strand awaits the
introduction of methyl groups, the two strands can be distinguished
• This provides the basis for a system to correct replication errors
• The dam gene codes for a methylase whose target is the adenine GATC
• The hemimethylated state is used to distinguish replicated origins from
nonreplicated origins
• The same target sites are used by a replication-related mismatch repair
system
 Mismatch errors occur
during replication in E. coli
• GATC sequences are targets for the Dam
methylase after replication
• During the period before this methylation
occurs
• The non methylated strand is the target for
repair of mismatched bases
 Recombination-Repair Systems in E. coli
• Use activities that overlap with those involved in genetic
recombination
• They are also sometimes called "post-replication repair" because
they function after replication
• Such systems are effective in dealing with the defects produced in
daughter duplexes by replication of a template that contains damaged
bases
 Recombination-Repair
Systems in E. coli
An E. coli retrieval system uses a normal
strand of DNA
To replace the gap left in a newly
synthesized strand
Opposite a site of unrepaired damage
 RecA Triggers the SOS System
• Bacteria engage in a more global response to damage the SOS
response
• Response depends on the recombination protein RecA
• RecA: activated by many treatments that damage DNA or inhibit
replication in E. coli
• Causes it to trigger the SOS response: complex series of phenotypic
changes involves the gene expression (products include repair
functions)
• Dual activities of the RecA protein make it difficult to know whether a
deficiency in repair in recA mutant cells is due to:
• loss of the DNA strand-exchange function of RecA
• some other function whose induction depends on the protease activity
 Activation of RecA
• LexA is a small (22 kD) protein
• Relatively stable in untreated cells
• Functions as a repressor at many operons

Regulatory circuits Target genes

 Induction of RecA
• Activation of RecA causes proteolytic cleavage of the product of the
lexA gene
• Causes LexA to undertake an autocatalytic cleavage
• Inactivates the LexA repressor function
• Coordinately induces all the operons to which it was bound