Differential Requirement for Proliferating Cell Nuclear Antigen in 5′ and 3′ Nick-directed Excision in Human Mismatch Repair

• Published in: The Journal of biological chemistry Vol. 279; no. 17; pp. 16912 - 16917
• Main Authors: Guo, Shuangli, Presnell, Steven R., Yuan, Fenghua, Zhang, Yanbin, Gu, Liya, Li, Guo-Min
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23.04.2004
• Subjects: Adenosine Triphosphatases - physiology; Bacterial Proteins - physiology; Base Pair Mismatch; Base Sequence; Blotting, Southern; Cell Nucleus - metabolism; Cyclin-Dependent Kinase Inhibitor p21; Cyclins - metabolism; DNA - metabolism; DNA Repair; DNA, Complementary - metabolism; DNA-Binding Proteins - physiology; Dose-Response Relationship, Drug; Escherichia coli - metabolism; Escherichia coli Proteins; Glutathione Transferase - metabolism; HeLa Cells; Humans; Molecular Sequence Data; MutS DNA Mismatch-Binding Protein; Proliferating Cell Nuclear Antigen - metabolism; Proliferating Cell Nuclear Antigen - physiology; Protein Structure, Tertiary; Recombinant Fusion Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M313213200

Proliferating cell nuclear antigen (PCNA) is involved in mammalian mismatch repair at a step prior to or at mismatch excision, but the molecular mechanism of this process is not fully understood. To examine the role of PCNA in mismatch-provoked and nick-directed excision, orientation-specific mismatch removal of heteroduplexes with a pre-existing nick was monitored in human nuclear extracts supplemented with the PCNA inhibitor protein p21. We show here that, whereas 3′ nick-directed mismatch excision was completely inhibited by low concentrations of p21 or a p21 C-terminal fusion protein, 5′ nick-directed excision was only partially blocked under the same conditions. No further reduction of the 5′ excision was detected when a much higher concentration of p21 C-terminal protein was used. These results suggest the following. (i) There is a differential requirement for PCNA in 3′ and 5′ nick-directed excision; and (ii) 5′ nick-directed excision is conducted by a manner either dependent on or independent of PCNA. Our in vitro reconstitution experiments indeed identified a 5′ nick-directed excision pathway that is dependent on PCNA, hMutSα, and a partially purified fraction from a HeLa nuclear extract.