Download Mismatch repair (MMR)- Correction of mismatched nucleotides and

Mismatch repair (MMR)Correction of mismatched nucleotides and small loops
The mechanism of mismatch repair has been studied most
thoroughly in E. coli. Several research groups have re-constituted
the repair process from purified proteins.
The proteins that initiate the repair process in E. coli are MutS,
MutL, and MutH.
1
How do mismatches arise?
Most mismatches are due to replication errors.
However, mismatches can also be produced by other
mechanisms--for example, by deamination of 5-methyl cytosine to
produce thymidine (T) improperly paired to G.
Regardless of the mechanism by which they are produced,
mismatches can always be repaired by the mismatch repair
pathway.
In cases where the appropriate DNA-N-glycosylase is available,
mismatches can also be repaired by the base excision repair
pathway.
The previous systems recognized DNA damage caused by
mutagens. They search for abnormal chemical structures, CPDs,
crosslinks.
BUT – they can not correct mismatches resulting from replication
errors because the mismatched nucleotide is not abnormal in any
way – it is simply A,T, C or G inserted in a wrong place.
MMR system that corrects replication errors detects not
mispaired nucleotides itself, but the absence of base-pairing
between the daughter and parental strand.
Once it wound the mismatch it will excise it and fill the gap.
2
Important question –
repair must be made on the daughter strand because it is in this
new synthesized strand that the error has occurred: the parent
strand has correct sequence.
How does the repair know which strand is which?
The answer:
In E. coli at this stage:
•daughter strand is undermethylated
•parent strand has a full complement of methyl groups.
Daughter strand is undermethylated
E. coli DNA is normally methylated at GATC sequences, but
the newly synthesized strand is not immediately methylated
since polymerases incorporate adenine, not methyladenine
into DNA.
The adenines on the daughter strand are methylated by a
specific enzyme Dam methyltransferase, only after a lag of
several minutes.
During this period a new strand contains hemimethylated
sequences.
3
Mismatch repair (MMR)
Mismatch repair (MMR) in E. coli
The replication-error-produced mismatch in the
above diagram is indicated by the distorted double
helix.
1. MutS protein recognizes such mismatches (true
mismatches plus insertions/deletions of up to 4
nucleotides) and binds to them. MutS activates
binding of MutL.
Scheme by Dr. Huberman
2. Binding of MutL stabilizes the complex. The
MutS-MutL complex activates MutH.
3. MutH which is able to distinguish hemimethylated sequences is thus able to distinguish a
new strand (presumably incorrect) from the
parental (presumably correct) strand. It locates a
nearby methyl group and nicks the newly
synthesized strand opposite the methyl group, as
MutH has endonuclease activity.
4
Mismatch repair (MMR) in E. coli
4. Next the segment of daughter strand containing misincorporated
base is excised and replaced with correct sequence.
Excision is accomplished by cooperation between
the UvrD (Helicase II) protein, which unwinds from the nick in
the direction of the mismatch,
and a single-strand specific exonuclease of appropriate polarity
(one of several in E. coli),
followed by resynthesis (Polymerase III)
and ligation (DNA ligase).
MMR in eukaryotes – proteins involved
There are several eukaryotic genes that appear to be
homologues of the corresponding E. coli MMR genes both in
terms of amino acid sequence and in terms of functional
similarities.
Whereas MutS and MutL function as monomers, the eukaryotic
proteins function as heterodimers.
heterodimers.
Dimers of MutS homologues are responsible for initial
recognition of mismatches and small insertions/deletions.
Dimers of MutL homologues interact with the resulting
complex, as in E. coli.
5
MMR in eukaryotes – proteins involved
Two heterodimers of MutS homologues are found in human cells.
One of these dimers (MSH2/MSH6) is called hMutSalpha. It
preferentially recognizes single base mismatches and small (1-4
base) loops.
The second (MSH2/MSH3) is called hMutSbeta and primarily
recognizes loops of a similar size range.
It is important to note that these specificities are not absolute;
MutSalpha and MutSbeta are individually capable of recognizing
both single base mismatches and loops of various sizes.
MMR in eukaryotes – proteins involved
Three MutL homolog dimers are known:
One dimer consists of MLH1 and PMS1(yeast)/PMS2(human)
and is called hMutLalpha.
The second dimer consists of MLH1 and PMS1(human) and is
called hMutLbeta.
The third dimer consists of MLH1 and MLH3 and has not yet
been assigned a name.
hMutLalpha can function with hMutSalpha
MutSalpha and with
hMutSbeta.
hMutSbeta
The roles of the other two MutL dimers in MMR are not yet
well established.
6
Human mismatch repair proteins
MMR in eukaryotes – proteins involved
At least two nucleases, exonuclease 1 (5' to 3' on dsDNA
substrates) and Flap Endonuclease (FEN-1 or DNase IV; Rad27
in S. cerevisiae)
cerevisiae) appear to contribute to mismatch repair in
eukaryotic cells, just as exonucleases are thought to be
important for mismatch repair in prokaryotes.
The precise roles of these nucleases have not yet been clarified.
7
MMR in eukaryotes – proteins involved
In eukaryotic cells, several standard replication proteins are
needed for mismatch repair.
The "clamp" protein, PCNA (a cofactor for both polymerases delta
and epsilon), is required to stabilize the MutS and MutL
heterodimers at mismatch sites on DNA and is also required
during the DNA synthesis step of mismatch repair.
This DNA synthesis step also requires RPA (the eukaryotic singlestranded DNA-binding protein), Replication factor C (which loads
PCNA onto DNA molecules at primer termini) and DNA
polymerase delta.
Model for mismatch
repair
Mammalian MMR involves multi-member
families of the E. coli prototype factors MutS
and MutL.
Heterodimers of hMSH2/6 (hMutSalpha) focus
on mismatches and single-base loops
(stage I in the figure below, upper strand),
whereas hMSH2/3 dimers (hMutSbeta)
recognize insertion/deletion loops (II, lower
strand).
8
Model for mismatch
repair
Heterodimeric complexes of the hMutL-like
proteins hMLH1/hPMS2 (hMutLalpha )
and hMLH1/hPMS1 (hMutLbeta )
interact with MSH complexes and replication
factors.
Strand discrimination may be based on contact
with the nearby replication machinery.
Model for
mismatch
repair
A number of proteins are implicated in the
excision of the new strand past the mismatch
and resynthesis steps,
including pol δ/ε , RPA, PCNA, RFC,
exonuclease 1, and endonuclease FEN1 (II,
III).
MMR components also interact functionally
with NER and recombination.
9
Model for
mismatch
repair
Recent crystallographic studies have
revealed that
a MutS dimer detects the structural
instability of a heteroduplex
by kinking the DNA at the site of the
mismatch,
which is facilitated when base pairing is
affected.
Model for
mismatch repair
However, DNA damage with similar
characteristics,
such as that caused by alkylating agents and
intercalators,
may fool MutS, triggering erroneous or futile
MMR.
Intact MMR thus confers sensitivity,
and as several of these agents are used in
chemotherapy,
tumours may become resistant to them on the
basis of selection for defective MMR,
so confounding therapeutic strategies
10
Mismatch repair & Hereditary non-polyposis
non-polyposis
colorectal cancer
•
HNPCC – 2-10% of all colorectal cancer (this is at
least 10 fold higher than the FAP syndrome);
autosomal dominant inheritance
•
A group of 5 similar syndromes (HNPCC1-5)
caused by mutations in the mismatch repair genes;
most mutations are in MLH1 and MSH2
•
Males heterozygous for mutant HNPCC gene have
~90% lifetime risk of developing colorectal cancer;
females have ~70% lifetime risk but also have
~40% risk for endometrial cancer
Microsatellite Instability (MIN or MSI, or
replication error positive or RER+)
• microsatellites: repetitive genetic loci (typically
1-5 bases, repeated 15-30 times)
• prone to ‘slipping’ during DNA replication →
insertions, deletions, normally repaired
- mutator phenotype occurs in HNPCC cases
• PCR-tested in cancer patients: occurs in 10+%
cases of sporadic colorectal cancer
•occurs in >90% in tumors from HNPCC family
members
11
Mutator Phenotype following loss of
mismatch repair
•
Loss of one allele does not impair mismatch repair
•
Inactivation of remaining allele (usually allele loss)
causes cell to acquire “mutator phenotype”
phenotype”
•
RER+ -replication error positive phenotype
•
hundreds of errors arise with each round of cell
replication and fail to be recognized and repaired –
subset of these are likely to activate oncogenes and
inactivate tumor suppressors
DNA Mismatch Repair
Repair of Replication Errors
Mechanisms for Insuring Replicative Fidelity
1. Base pairing
2. DNA polymerases
- base selection
- proofreading
3. Accessory proteins
- single strand binding protein
4. Mismatch correction
10-1 to 10-2
10-5 to 10-6
10-7
10-10
12
Eucaryotic homologs of MMR genes
Germline mutations occur in the syndrome: Hereditary nonpolyposis colon cancer - HNPCC
Approx. 90% of MMR mutations occur in Msh2 and Mlh1
HNPCC accounts for approx. 3% of all colon cancers
Mismatch Repair Mutations in
Hereditary Nonpolyposis Colon Cancer
(HNPCC)
• MMR mutations in 70% of families
• MLH1 (50%), MSH2 (40%)
• Minor role for MSH6, PMS1, PMS2
• Population prevalence 1:2851 (15-74 years)
• 18% of colorectal cancers under 45 years
• 28% of colorectal cancers under 30 years
13
Functions of MMR Proteins
 Repair of mismatches and insertion/deletion loops
- Msh2, Msh3, Msh6, Mlh1, Pms2, (Pms1, Mlh3)
 Meiotic recombination
- Msh4, Msh5, Mlh1, Pms2, Mlh3
 Mitotic recombination
- Msh2, Msh3
 DNA damage signaling in apoptosis (alkylation damage)
- Msh2, Msh6, Mlh1, Pms2
 Repair of DNA Interstrand Cross-links
- Msh2, Msh3, Mlh1?, Pms2?
Interactions in Mammalian MMR
Up to about 12 nucleoti des
Msh2/Msh6
MutSα (recognizes base-base mismatch and 1bp IDL)
Msh2/Msh3
MutSβ (recognizes 2 to approx. 12 bp IDLs)
Mlh1/Pms2
MutLα
Mlh1/Pms1
MutLβ
Mlh1/Mlh3
14
MMR Genes in Colorectal Neoplasia
Population incidence
MMR deficiency prevalencea
MMR gene mutations
hMSH2
hMLH1
hPMS2
Nature of Mutations b
Truncating
Missense
HNPCC
Sporadic Cancers
~ 1 in 500
> 90% of kindreds c
> 70% f
45% f
49% f
6% f
1 in 20
13% e
~ 65% of CRC with MIg
60% h
35% h
5% h
70% f
30% f
55% h
45% h
MMR = Mismatch Repair
CRC = Colorectal Cancer
a
As assessed by presence o microsatellite instability (MI).
b
Based on MMR mutation that could be precisely defined at the nucleotide level. For the purpose of this
table, frameshift, nonsense, and splice site mutations as well as large intragenic deletions were considered
“truncating.” Three basepair deletions were counted as missense mutations.
f
Based on 33 mutations in 47 kindreds. In addition, a hPMS1 mutation was identified in a single kindred
(Liu et al., 1996)
g
Based on 15 cases published as of September 1, 1996.
h
Based on 20 somatic mutations published as of September 1, 1996.
Multi-step pathway for development of
sporadic colon cancer
Genes responsible for HNPCC and FAP are involved in sporadic
colon cancer.
15
HNPCC – disease associated with MMR defficiency
References:
1. http://www.web-books.com/MoBio/Free/Ch10D.htm
2. M. Esteller et al., DNA methylation patterns in hereditary human
cancer mimic sporadic tumorigenesis. (2001) Hum. Mol. Genet. 10,
3001-3007.
3. Lodish et al., Molecular Cell Biology, Freeman and Co.
4. E. Evans and E. Alani, Roles for Mismatch Repair Factors in
Regulating Genetic Recombination. (2000) Molecular and Cellular
Biology, 20, 7839-7844.
5. B. Vogelstein and K. Kinzler, 1993, Trends Genet. 9:101
16
Literature sources:
T.A. Brown. Genomes, John Wiley and Sons,Inc., New-York,p. 330350 (1999).
E.Friedberg, G. Walker, W. Siede. DNA repair and mutagenesis,
ASM press, Washington DC, 1995
B. Lewin. Genes VII, Oxford University Press.
J. Huberman (2001) DNA repair. Roswell Park Cancer Institute.
R. Weaver, Molecular Biology, 2002, McGraw Hill
Hoeijmakers, J. Genome maintenance mechanisms for preventing
cancer. Nature 411, 366-374 (2001).
17