DNA Mismatch Repair
MutS and MutL
interaction with the beta sliding clamp
Reference: Lopez de Saro FJ, Marinus MG, Modrich P, O'Donnell M. (2006)
The beta
sliding clamp binds to multiple sites within MutL and MutS. J Biol
Chem. 281, 14340-9.
The MutL and MutS proteins are the central components of the DNArepair
machinery that corrects mismatches generated byDNA polymerases during
synthesis. We find that MutL interacts directly with the beta sliding
clamp, a ring-shaped dimeric protein that confers processivity to DNA
polymerases by tethering them to their substrates. Interestingly, the
interaction of MutL with beta only occurs in the presence of
single-stranded DNA. We find that the interaction occurs via a loop in
MutL near the ATP-binding site. The binding site of MutL on beta
locates to the hydrophobic pocket between domains two and three of the
clamp. Site-specific replacement of two residues in MutL diminished
interaction with beta without disrupting MutL function with helicase
II. In vivo studies reveal that this mutant MutL is no longer
functional in mismatch repair. In addition, the human MLH1 has a close
match to the proliferating cell nuclear antigen clamp binding motif in
the region that corresponds to the beta interaction site in Escherichia coli MutL, and a
peptide corresponding to this site binds proliferating cell nuclear
antigen. The current report also examines in detail the interaction of
with MutS. We find that two distinct regions of MutS interact
with.beta One is located near the C terminus and the other is
close to the N terminus, within the mismatch binding domain.
Complementation studies using genes encoding different MutS mutants
reveal that the N-terminal beta interaction motif on MutS is
essential for activity n ivivo, but the C-terminal interaction site for
is not. In light of these results, we propose roles for the beta
clamp in orchestrating the sequence of events that lead to mismatch
repair in the cell.
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The MutS
C-terminus
Reference: Calmann, M.A., Nowosielska, A. and Marinus, M.G. (2005) The
MutS
C-terminus is essential for mismatch repair activity in vivo. J.
Bacteriol. 2005 187: 6577-6579.
An Escherichia coli K-12
strain was constructed with a chromosomal deletion (mutS800) in the mutS gene that produced the removal
of the C-terminal 53 amino acids which are not present in the MutS
crystal structure. This strain has a MutS null phenotype for mutation
avoidance, antirecombination, and sensitivity to cytotoxic agents in a dam mutant background.
MutS split-phenotype
Reference: Calmann, M.A., Nowosielska, A. and Marinus, M.G. (2005)
Separation of mutation avoidance and antirecombination functions in an Escherichia coli mutS mutant.
Nucleic
Acids Research 33, 1193-1200.
DNA mismatch repair in Escherichia
coli has been shown to be involved in two distinct processes:
mutation avoidance, which removes potential mutations arising as
replication errors, and antirecombination which prevents recombination
between related, but not identical (homeologous), DNA sequences. We
show that cells with the mutS800
mutation (which removes the C-terminal 53 amino acids of MutS) on a
multicopy plasmid are proficient for mutation avoidance. In
interspecies genetic crosses, however, recipients with the mutS800 mutation show increased
recombination by up to 280-fold relative to mutS. The MutSD800 protein binds to
O6-methylguanine mismatches but not to intrastrand platinated GG
cross-links, explaining why dam
bacteria with the mutS800
mutation are resistant to cisplatin, but not MNNG, toxicity. The
results indicate that the C-terminal end of MutS is necessary for
antirecombination and cisplatin sensitization, but less significant for
mutation avoidance. The inability of MutS800 to form tetramers may
indicate that these are the active form of MutS.
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