Structure-Function Studies of the Initiation Response of Human Mismatch Repair Proteins to DNA Containing a Mismatch Public Deposited

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  • March 19, 2019
  • Bradford, Kira
    • Affiliation: College of Arts and Sciences, Department of Chemistry
  • DNA mismatch repair (MMR) is the post-replicative process that recognizes and repairs misincorporated bases that occur during replication. Deficiencies in MMR are linked to greater than 80% of hereditary non-polyposis colorectal cancer. To better understand the initiation of MMR in humans, two proteins, MSH2-MSH6 (hMutSα) and MLH1-PMS2 (hMutLα), were studied. hMutSα first recognizes and binds to the mismatch in an ADP-dependent manner; however, it then undergoes an ATP-dependent conformational change(s) into a mobile clamp. The ATP activated form of hMutSα also recruits hMutLα to the DNA. Together, these proteins signal the downstream events of MMR that results in the repair of the misincorporated base. Currently, the conformations of the hMutSα-DNA complexes in the presence of ATP are not well characterized. hMutSα in the presence of ADP has previously been crystallized in complex with mismatched DNA. This crystal structure shows that hMutSα induces a kink on the DNA, consistent with other studies done with MutS homologs. However, no crystal structures have yet been solved of hMutSα in the presence of ATP. Additionally, the conformational state of hMutSα that interacts with hMutLα is not well understood, and little is known on the conformations of the hMutSα-hMutLα-DNA complexes. iv In this work, I used atomic force microscopy (AFM) to examine the conformational properties of hMutSα and hMutSα-hMutLα on DNA containing a single mismatch and in various adenine nucleotide conditions. The data suggests that hMutSα recognizes and binds to the mismatch in the presence of ADP, and, surprisingly, remains localized to the mismatch in the presence of ATP. The data also show that ATP induces hMutSα-hMutSα interactions, and multiple hMutSα form a complex on the mismatch. I also characterized the hMutSα-induced DNA bend angle properties and saw unique changes in bending depending on the adenine nucleotide conditions. Additionally, I observed unique properties of the complexes of hMutSα and hMutLα in the presence of ATP including: complex volumes becoming larger, DNA lengths appearing shorter, and some population of these complexes remain localized to the mismatch. Taken together, these data provide mechanistic insights and allows us to propose a molecular model for MMR initiation.
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  • In Copyright
  • Erie, Dorothy
  • Spremulli, Linda
  • Ramsden, Dale
  • Kunkel, Tom
  • Redinbo, Matthew R.
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2015
Place of publication
  • Chapel Hill, NC
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