Related Experiment Videos
Transfer of the MSH2.MSH6 complex from proliferating cell nuclear antigen to mispaired bases in DNA
Patrick J Lau1, Richard D Kolodner
1Ludwig Institute for Cancer Research, Cancer Center and Department of Medicine, University of California San Diego School of Medicine, La Jolla, California 92093-0660, USA.
Abstract:
Proliferating cell nuclear antigen (PCNA) is thought to play a role in DNA mismatch repair at the DNA synthesis step as well as in an earlier step. Studies showing that PCNA interacts with mispair-binding protein complexes, MSH2.MSH3 and MSH2.MSH6, and that PCNA enhances MSH2.MSH6 mispair binding specificity suggest PCNA may be involved in mispair recognition. Here we show that PCNA and MSH2.MSH6 form a stable ternary complex with a homoduplex (G/C) DNA, but MSH2.MSH6 binding to a heteroduplex (G/T) DNA disrupts MSH2.MSH6 binding to PCNA. We also found that the addition of ATP or adenosine 5'-O-(thiotriphosphate) restores MSH2.MSH6 binding to PCNA, presumably by disrupting MSH2.MSH6 binding to the heteroduplex (G/T) DNA. These results support a model in which MSH2.MSH6 binds to PCNA loaded on newly replicated DNA and is transferred from PCNA to mispaired bases in DNA.
Insights
Proliferating cell nuclear antigen (PCNA) may recognize DNA mismatches by interacting with MSH2.MSH6. ATP disrupts this interaction, facilitating MSH2.MSH6 transfer to mismatched DNA bases.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Protein-DNA Interactions
Background:
- Proliferating cell nuclear antigen (PCNA) is implicated in DNA mismatch repair (MMR).
- PCNA's interaction with MSH2.MSH3 and MSH2.MSH6 complexes suggests a role in mispair recognition.
- PCNA enhances the specificity of MSH2.MSH6 binding to mismatched DNA.
Purpose of the Study:
- To investigate the role of PCNA in DNA mismatch recognition by MSH2.MSH6.
- To elucidate the mechanism of PCNA-MSH2.MSH6 interaction during DNA repair.
Main Methods:
- Formation of ternary complexes involving PCNA, MSH2.MSH6, and DNA duplexes (homoduplex G/C and heteroduplex G/T).
- Assessing the effect of ATP and its analog on these complex formations.
- Investigating the disruption of MSH2.MSH6 binding to PCNA upon binding to heteroduplex DNA.
Main Results:
- PCNA and MSH2.MSH6 form a stable ternary complex with homoduplex DNA.
- MSH2.MSH6 binding to heteroduplex DNA disrupts its interaction with PCNA.
- ATP or adenosine 5'-O-(thiotriphosphate) addition restores PCNA-MSH2.MSH6 binding, likely by disrupting MSH2.MSH6 binding to heteroduplex DNA.
Conclusions:
- These findings support a model where MSH2.MSH6 is transferred from PCNA to mismatched DNA bases.
- PCNA acts as a platform for MSH2.MSH6 loading onto newly replicated DNA.
- The dynamic interaction between PCNA and MSH2.MSH6 is crucial for efficient DNA mismatch recognition.