Methyldirected mismatch repair in E. coli:

1
Methyl-directed mismatch repair in E. coli

• MutS protein recognizes and binds to the
  mismatch
• MutL helps to recruit MutH, which recognizes and
  cleaves one DNA strand (the strand that is not
  methylated)
• an exonuclease resects the DNA past the mismatch
• DNA polymerase (III) fills in the gap using the
  intact strand as the template
• DNA ligase (not shown) seals the nick

The adenine in GATC sequences in E. coli and
other Gram-negative enteric bacteria is
methylated on N6. Immediately after replication,
the newly synthesized strand is briefly
unmethylated, which signals that this strand
should be repaired. This elegant mechanism for
distinguishing old from new DNA is not used
by most organisms
c.f. Fig. 20.39
2
Mismatch Repair in humans
- no MutH homologs, and mechanism of strand
recognition is still unclear.
? Mutations in Mlh1, Msh2, and Msh6 are
associated with hereditary nonpolyposis
colorectal cancer (HNPCC). Overall, mutations in
these genes cause 13 of all colorectal,
endometrial, and gastric cancers.

• Patients with HNPCC are heterozygous for the
  mutations and have reduced mismatch repair, as
  well as increased microsatellite instability
• tumors from the patients have lost the wild-type
  allele
• it is not known why defects in mismatch repair
  specifically cause colon cancer

3
Repair of damaged bases and DNA adducts (Direct
reversal and Excision Repair)
Damaged bases are caused by - spontaneous
deamination

• or are chemically induced (nitrous acid,
  bisulfites..)

Friedberg, DNA Repair
4
Mutagenic consequences of C ? U deamination
Lodish, p.476
5
(No Transcript)
6
Chemically-induced adducts
example Benzo-a-pyrene (one of the primary
carcinogens in tar) is metabolized to a
nucleophilic form (most likely the
7,8-diol-9,10-epox form) which intercalates into
DNA helices and forms adducts with the NH2 group
of guanine.
7

• Direct reversal corrects DNA adducts directly
  (without replication fill-in)

photoreactivation catalyzed by photolyase breaks
intra-strand crosslinks in pyrimidine dimers, but
not 6-4 photoproducts. Present in almost
organisms except, unfortunately, mammals.
O6-methylguanine methyl transferase catalyzes the
removal of alkyl groups from the O6 position of
guanine the methyl group is transferred to a
cysteine residue on the enzyme, inactivating it
("suicide enzyme") The methylated form of the
enzyme functions as a transcription factor in
activating txn of its own operon
Fig. 20.32
Fig. 20.31
8

• Excision repair
• removes damaged bases and DNA adducts and
  fills in gap using intact strand as template

separated into Base Excision Repair, "BER"
(removal of a damaged base, usually not involving
severe distortion of the helix) and Nucleotide
Excision Repair "NER" (removal of many
nucleotides including the damaged base or adduct)
BER

• DNA glycosylase recognizes the damaged base,
  cuts the bond linking the base to the deoxyribose
• AP endonuclease cuts the sugar-phosphate
  backbone on the 5' side of the position lacking
  the base (AP site)
• DNA phosphodiesterase removes the deoxyribose
  phosphate that originally was connected to the
  damaged base

• spontaneous base loss also fixed by this
  mechanism (AP endo, etc)

Fig. 20.33
9
BER is similar in eukaryotes, except that the
major repair DNAP, Pol?, directly removes the
5ribose phosphate
Fig. 20.34
10
DNA glycosylases are the critical recognition
component of BER. Organisms contain many
different glycosylases, each recognizing a
different damaged base. One of the most important
is Uracil DNA glycosylase, which removes uracil
misincorporated into DNA.
E. coli and yeast cells deficient in some of the
BER enzymes show elevated rates of spontaneous
mutation. Thus, alkylating agents may be present
in vivo as products of normal metabolism.
11
Nucleotide excision repair (NER) removal of
bulky adducts and intra-strand crosslinks by
excision of the DNA strand containing the adduct
NER in E. coli

• UvrAB complex translocates along DNA, using the
  energy of ATP hydrolysis
• The complex pauses at a damage site (distorted
  helix) and UvrA is released
• UvrC is recruited to the UvrB-DNA complex and
  nicks the damaged strand, 12-13 nt apart

Fig. 20.36
12
mechanistically similar to prokaryotic NER in
damage recognition, unwinding of helix
surrounding the damage site, and dual incisions
NER in humans

• Several of the genes now known to be involved in
  NER were first identified by complementation of
  rodent and human cell lines deficient in repair

• components of the general transcription factor
  complex TFIIH (XPB and XPD) are integral to the
  NER process in eukaryotes. These also link NER to
  trancriptional-coupled repair (TCR)...

Fig. 20.37
13
Transcription-Coupled Repair NER is directed
preferentially at transcribed strands

• RNA polymerase acts as a sensor of DNA damage
  during transcription

preferential repair of genomic regions that are
actively transcribed
preferential repair of the transcribed strand
over the non-transcribed strand (requires active
RNAP II)
14
Hereditary Disorders associated with NER
Xeroderma Pigmentosum (XP) rare autosomal
recessive disorder - extreme sensitivity to
DNA-damaging agents, particularly UV
radiation - skin cancer at a young age - cells
defective in NER - neurological disorders
Cockayne's Syndrome (CS) rare autosomal
recessive disorder - extreme sensitivity to
DNA-damaging agents, particularly UV
radiation - congenital abnormalities, fatal in
young adulthood - dwarfism - neurological
disorders - defects in NER but specifically in
TCR
Some complementation groups of CS are allelic to
XP variants, but not all
15
(No Transcript)
16

• Homologous Recombination

Eukaryotic homologous recombination
E. coli
Several factors required in E. coli have homologs
in human cells E. coli recombinase RecA
Rad51 in eukaryotes (plus Rad51 homologs Rad55,
Rad57) single-strand binding protein SSB RPA,
No RecBCD in eukaryotes, but function probably
equivalent to the Mre11/Rad50/Nbs1 complex
(MRN) still unclear what the eukaryotic Holliday
junction resolvase is
Also implicated in homologous forms of
double-strand break repair are Brca1 and Brca2,
which were identified as strong hereditary risk
factors for breast and ovarian cancer. Brca2
binds to and perhaps regulates Rad51.