Investigating DNA remodeling in DNA mismatch repair

Many DNA metabolic pathways, including DNA repair, require the transmission of signals across long stretches of DNA or between DNA molecules. Solutions to this signaling challenge involve various mechanisms: protein factors can travel between these sites, loop DNA between sites, or form oligomers that bridge the spatial gaps. How these paradigms have been used to explain DNA mismatch repair which involves several steps that require action-at-a-distance has not been previously explained. In this thesis, we describe these models in detail and how current data fits into these descriptions. We also outline regulation steps that remain unanswered in how action is communicated across long distances along a DNA contour in DNA mismatch repair. In eukaryotes, mismatch repair begins with MutS homolog (MSH) complexes detecting mismatches and recruiting the processivity clamp (PCNA)-stimulated endonuclease Mlh1-Pms1/PMS2 (yeast/human), which nicks the DNA distal to the mismatch location and a pre-existing single strand break, that may allow the pathway to distinguish between DNA strands. The Mlh1-Pms1/PMS2-generated nick allows downstream proteins to remove the mismatch. How the Mlh1-Pms1/PMS2 endonuclease operates across lengths of DNA to facilitate nicking DNA is critical for mismatch repair, but presents a key signaling challenge to the pathway. Using purified yeast proteins, we show that Mlh1-Pms1 uses ATP to compact DNA, a behavior which may act as a search mechanism for strand-discrimination signals, which may be hundreds of base pairs away from the mismatch and the site where Mlh1-Pms1/PMS2 nicks. When a pre-existing nick is encountered, compaction is suppressed and Mlh1-Pms1 instead stabilizes the site, protecting it from melting induced by PCNA’s loader, RFC. Phased nicking assays further reveal that the timing of Mlh1-Pms1 encountering a pre-existing nick relative to RFC/PCNA determines whether the complex remains suppressed or becomes activated. Together, these results support a model of Mlh1-Pms1 using ATP-driven global compaction to toggle between a search mode and a PCNA-licensed repair mode, ultimately facilitating repair across distances of DNA. In this thesis, we also investigate how the intrinsically disordered regions in Mlh1-Pms1/PMS2 contribute to activation and recycling of the endonuclease during DNA mismatch repair. Using previously identified point mutations within the intrinsically disordered region of yeast Mlh1 (R401A, D403A), which are conserved in the human protein, we determined that Mlh1’s intrinsically disordered region contributes to iterative nicking by Mlh1-Pms1. Using assays to determine the number of nicks Mlh1-Pms1 makes on the DNA, we identified a lack of reactivation following one incision in the DNA when R401 and D403 were substituted with alanine. Exonuclease protection assays uncovered that mutations to the intrinsically disordered region of Mlh1 prevent nick disengagement by Mlh1-Pms1 following one round of endonuclease activation. These results support a model of nick disengagement by Mlh1-Pms1 mediated by the intrinsically disordered regions, followed by subsequent reactivation to product iterative nicks within the DNA. Importantly, these findings establish the intrinsically disordered regions as key regulators of endonuclease turnover rather than catalytic activity itself, revealing a previously underappreciated layer of control that is likely essential for coordinating efficient mismatch removal and maintaining genome stability.

Grantor dc:publisher

Temple University. Libraries

Year dc:date.issued

2026

Author dc:creator

• Collingwood, Bryce William

Advisor dc:contributor.advisor

• Manhart, Carol

Committee members dc:contributor.committeemember

• Valentine, Ann M.
• Wang, Rongsheng
• Balakrishnan, Lata

• Biochemistry
• DNA repair
• Mismatch repair
• Mlh1-Pms1
• Chemistry

Statement dc:rights

• IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available.

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

eng

Repository record dc:identifier.uri

https://scholarshare.temple.edu/handle/20.500.12613/12218

OAI identifier oai:identifier

oai:scholarshare.temple.edu:20.500.12613/12218