scholarly journals Recurrent mismatch binding by MutS mobile clamps on DNA localizes repair complexes nearby

2020 ◽  
Vol 117 (30) ◽  
pp. 17775-17784 ◽  
Author(s):  
Pengyu Hao ◽  
Sharonda J. LeBlanc ◽  
Brandon C. Case ◽  
Timothy C. Elston ◽  
Manju M. Hingorani ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Residence Time ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Dna Mismatch ◽  
Single Molecule Fret ◽  
Current Thinking ◽  
Mismatch Recognition ◽  
The Guardian ◽  
The Impact

DNA mismatch repair (MMR), the guardian of the genome, commences when MutS identifies a mismatch and recruits MutL to nick the error-containing strand, allowing excision and DNA resynthesis. Dominant MMR models posit that after mismatch recognition, ATP converts MutS to a hydrolysis-independent, diffusive mobile clamp that no longer recognizes the mismatch. Little is known about the postrecognition MutS mobile clamp and its interactions with MutL. Two disparate frameworks have been proposed: One in which MutS–MutL complexes remain mobile on the DNA, and one in which MutL stops MutS movement. Here we use single-molecule FRET to follow the postrecognition states of MutS and the impact of MutL on its properties. In contrast to current thinking, we find that after the initial mobile clamp formation event, MutS undergoes frequent cycles of mismatch rebinding and mobile clamp reformation without releasing DNA. Notably, ATP hydrolysis is required to alter the conformation of MutS such that it can recognize the mismatch again instead of bypassing it; thus, ATP hydrolysis licenses the MutS mobile clamp to rebind the mismatch. Moreover, interaction with MutL can both trap MutS at the mismatch en route to mobile clamp formation and stop movement of the mobile clamp on DNA. MutS’s frequent rebinding of the mismatch, which increases its residence time in the vicinity of the mismatch, coupled with MutL’s ability to trap MutS, should increase the probability that MutS–MutL MMR initiation complexes localize near the mismatch.

scholarly journals Toward Single Molecule FRET Studies of DNA Mismatch Repair in Live Bacteria

2019 ◽  
Vol 116 (3) ◽  
pp. 139a
Author(s):  
Pengning Xu ◽  
Andrew Hensley ◽  
Edward Chan ◽  
Keith R. Weninger
Keyword(s):  
Mismatch Repair ◽  
Single Molecule ◽  
Dna Mismatch Repair ◽  
Dna Mismatch ◽  
Single Molecule Fret ◽  
Live Bacteria

scholarly journals MutL traps MutS at a DNA mismatch

2015 ◽  
Vol 112 (35) ◽  
pp. 10914-10919 ◽  
Author(s):  
Ruoyi Qiu ◽  
Miho Sakato ◽  
Elizabeth J. Sacho ◽  
Hunter Wilkins ◽  
Xingdong Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Thermus Aquaticus ◽  
Dna Mismatch ◽  
Sliding Clamp ◽  
Single Molecule Fret ◽  
Mismatch Detection ◽  
Current Thinking ◽  
Processivity Factor ◽  
Subsequent Activation

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL’s endonuclease activity by β-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS–MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with β-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.

scholarly journals Toward Adding Complexity in Single Molecule FRET Studies of DNA Mismatch Repair

2015 ◽  
Vol 108 (2) ◽  
pp. 69a-70a
Author(s):  
keith weninger ◽  
Pengyu Hao ◽  
Yue Yang ◽  
Elizabeth J. Sacho ◽  
Ruoyi Qiu
Keyword(s):  
Mismatch Repair ◽  
Single Molecule ◽  
Dna Mismatch Repair ◽  
Dna Mismatch ◽  
Single Molecule Fret

Induction of recombination between homologous and diverged DNAs by double-strand gaps and breaks and role of mismatch repair

1994 ◽  
Vol 14 (7) ◽  
pp. 4802-4814
Author(s):  
S D Priebe ◽  
J Westmoreland ◽  
T Nilsson-Tillgren ◽  
M A Resnick
Keyword(s):  
Gene Conversion ◽  
Mismatch Repair ◽  
Sequence Divergence ◽  
Strand Break ◽  
Mismatch Repair Gene ◽  
Repair Gene ◽  
Heteroduplex Dna ◽  
Dna Mismatch ◽  
Spontaneous Recombination ◽  
The Impact

Sequence homology is expected to influence recombination. To further understand mechanisms of recombination and the impact of reduced homology, we examined recombination during transformation between plasmid-borne DNA flanking a double-strand break (DSB) or gap and its chromosomal homolog. Previous reports have concentrated on spontaneous recombination or initiation by undefined lesions. Sequence divergence of approximately 16% reduced transformation frequencies by at least 10-fold. Gene conversion patterns associated with double-strand gap repair of episomal plasmids or with plasmid integration were analyzed by restriction endonuclease mapping and DNA sequencing. For episomal plasmids carrying homeologous DNA, at least one input end was always preserved beyond 10 bp, whereas for plasmids carrying homologous DNA, both input ends were converted beyond 80 bp in 60% of the transformants. The system allowed the recovery of transformants carrying mixtures of recombinant molecules that might arise if heteroduplex DNA--a presumed recombination intermediate--escapes mismatch repair. Gene conversion involving homologous DNAs frequently involved DNA mismatch repair, directed to a broken strand. A mutation in the PMS1 mismatch repair gene significantly increased the fraction of transformants carrying a mixture of plasmids for homologous DNAs, indicating that PMS1 can participate in DSB-initiated recombination. Since nearly all transformants involving homeologous DNAs carried a single recombinant plasmid in both Pms+ and Pms- strains, stable heteroduplex DNA appears less likely than for homologous DNAs. Regardless of homology, gene conversion does not appear to occur by nucleolytic expansion of a DSB to a gap prior to recombination. The results with homeologous DNAs are consistent with a recombinational repair model that we propose does not require the formation of stable heteroduplex DNA but instead involves other homology-dependent interactions that allow recombination-dependent DNA synthesis.

scholarly journals Phosphorylation of PCNA by EGFR inhibits mismatch repair and promotes misincorporation during DNA synthesis

2015 ◽  
Vol 112 (18) ◽  
pp. 5667-5672 ◽  
Author(s):  
Janice Ortega ◽  
Jessie Y. Li ◽  
Sanghee Lee ◽  
Dan Tong ◽  
Liya Gu ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Mismatch Repair ◽  
Posttranslational Modifications ◽  
Growth Factor Receptor ◽  
Nuclear Antigen ◽  
Dna Mismatch ◽  
Epidermal Growth ◽  
Mismatch Recognition ◽  
Proliferating Cell

Proliferating cell nuclear antigen (PCNA) plays essential roles in eukaryotic cells during DNA replication, DNA mismatch repair (MMR), and other events at the replication fork. Earlier studies show that PCNA is regulated by posttranslational modifications, including phosphorylation of tyrosine 211 (Y211) by the epidermal growth factor receptor (EGFR). However, the functional significance of Y211-phosphorylated PCNA remains unknown. Here, we show that PCNA phosphorylation by EGFR alters its interaction with mismatch-recognition proteins MutSα and MutSβ and interferes with PCNA-dependent activation of MutLα endonuclease, thereby inhibiting MMR at the initiation step. Evidence is also provided that Y211-phosphorylated PCNA induces nucleotide misincorporation during DNA synthesis. These findings reveal a novel mechanism by which Y211-phosphorylated PCNA promotes cancer development and progression via facilitating error-prone DNA replication and suppressing the MMR function.

scholarly journals Functional Studies on the Candidate ATPase Domains of Saccharomyces cerevisiae MutLα

2000 ◽  
Vol 20 (17) ◽  
pp. 6390-6398 ◽  
Author(s):  
Phuoc T. Tran ◽  
R. Michael Liskay
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Dna Mismatch Repair ◽  
Limited Proteolysis ◽  
Atp Binding ◽  
Dna Mismatch ◽  
Functional Studies

ABSTRACT Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer, termed MutLα, that is required for DNA mismatch repair after mismatch binding by MutS homologues. Recent sequence and structural studies have placed the NH2 termini of MutL homologues in a new family of ATPases. To address the functional significance of this putative ATPase activity in MutLα, we mutated conserved motifs for ATP hydrolysis and ATP binding in both Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repair in vivo. Limited proteolysis with purified recombinant MutLα demonstrated that the NH2 terminus of MutLα undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs. Furthermore, two-hybrid analysis suggested that these ATP-binding-induced conformational changes promote an interaction between the NH2 termini of Mlh1p and Pms1p. Surprisingly, analysis of specific mutants suggested differential requirements for the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair. Taken together, these results suggest that MutLα undergoes ATP-dependent conformational changes that may serve to coordinate downstream events during yeast DNA mismatch repair.

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