In E. coli, genomic stability is maintained by a methyl-directed mismatch repair (MMR) pathway which reduces errors such as mismatched base-pairs or nucleotide insertions/deletions in newly- replicated DNA molecules by 1000-fold. Defects in the homologues of E. coli MMR proteins in humans are associated with increased rates of cancer development, and as E. coli MMR is among the best-studied DNA repair pathways, elucidation of its molecular mechanisms promises to shed new light into mutation avoidance within the cell. The MMR pathway is initiated when complexes of protein MutS identify and bind to a replication error (RE) on a newly-replicated DNA molecule and, with protein MutL, activate endonuclease MutH. MutH then nicks the newly-replicated strand at a hemi-methylated d(GATC) site that serves to discriminate between the new and original strands. This site can be over 1000 base-pairs away with no apparent directional bias (5'- or 3'- from the RE), but 3'-to-5'helicase UvrD is then loaded at the nick toward the RE and with the appropriate exonuclease removes the newly-replicated strand through the RE to be re- synthesized correctly. While the roles of individual MMR-associated proteins (MAPs) are well- established, how different MAP complexes efficiently coordinate the GATC-to-RE excision across large spans of DNA is still the subject of debate. Under physiological conditions MutS exists in an equilibrium of dimeric and tetrameric complexes, the latter having been recently observed on ''looped''DNA molecules with REs;although the role of MutS tetramers is disputed, the looping of DNA by MutS tetramers is proposed to be how a RE is 'coupled'to a distant d(GATC) site- that is, how a d(GATC) site near the RE can be efficiently found and how excision is limited to the DNA between the two sites. This hypothesis regarding the role of DNA looping by MutS tetramers will be investigated by the following specific aims: (1) To directly measure the sequence- and error-specific binding forces between DNA and MAP complexes as the pathway progresses. The search for REs then d(GATC) sites by MAP complexes will be investigated at the single-molecule (s.m.) level using force spectroscopic (FS) techniques and a nanotechnological FS apparatus. Combination with fluorescence microscopy will allow us to determine the roles of specific MAP complexes, resolve the effects of co-factors on their behavior, and map their spatio-temporal interactions along DNA. (2) To elucidate the mechanistic details of RE-to-d(GATC) ''coupling''and its relationship to DNA excision. Whether and how DNA looping alters the kinetics / efficiency of d(GATC) nicking, biased directional loading of UvrD, or excision termination will be addressed via atomic force microscopy, tethered particle motion experiments, and s.m. fluorescence assays.

Public Health Relevance

The proteins associated with the DNA mismatch repair (MMR) pathway in E. coli are responsible for ensuring that DNA molecules in the cell are copied accurately, and they do so by scanning along newly-made DNA molecules to identify then correct any errors in the sequence;very similar proteins have been found in nearly every known species, and in humans, defects in these proteins have been associated with increased rates of hereditary nonpolyposis colorectal cancer (Lynch syndrome) and spontaneous cancer development. While the roles of individual proteins in this pathway have long-since been identified, how these proteins interact with each other and how these interactions affect their abilities to correct errors in DNA are less well understood, but their elucidation remains important for understanding how DNA molecules are accurately copied inside living organisms. By developing new methods which will allow us to observe individual proteins associated with the MMR pathway-and record how these proteins scan along the DNA, identify errors, and manipulate the DNA molecules-we can determine the underlying mechanisms of mismatch repair and determine what ensures that errors in a DNA molecule are corrected quickly, selectively, and reliably in a cell.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1)
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Hoodbhoy, Tanya
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Duke University
Engineering (All Types)
Biomed Engr/Col Engr/Engr Sta
United States
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Josephs, Eric A; Marszalek, Piotr E (2018) Endonuclease-independent DNA mismatch repair processes on the lagging strand. DNA Repair (Amst) 68:41-49
Josephs, Eric A; Marszalek, Piotr E (2017) A 'Semi-Protected Oligonucleotide Recombination' Assay for DNA Mismatch Repair in vivo Suggests Different Modes of Repair for Lagging Strand Mismatches. Nucleic Acids Res 45:e63
Scholl, Zackary N; Josephs, Eric A; Marszalek, Piotr E (2016) Modular, Nondegenerate Polyprotein Scaffolds for Atomic Force Spectroscopy. Biomacromolecules 17:2502-5
Josephs, Eric A; Kocak, D Dewran; Fitzgibbon, Christopher J et al. (2015) Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage. Nucleic Acids Res 43:8924-41
Josephs, Eric A; Zheng, Tianli; Marszalek, Piotr E (2015) Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair. DNA Repair (Amst) 35:71-84