TY - JOUR
T1 - Differential Requirement for Proliferating Cell Nuclear Antigen in 5′ and 3′ Nick-directed Excision in Human Mismatch Repair
AU - Guo, Shuangli
AU - Presnell, Steven R.
AU - Yuan, Fenghua
AU - Zhang, Yanbin
AU - Gu, Liya
AU - Li, Guo Min
PY - 2004/4/23
Y1 - 2004/4/23
N2 - 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 hetero-duplexes 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.
AB - 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 hetero-duplexes 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.
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U2 - 10.1074/jbc.M313213200
DO - 10.1074/jbc.M313213200
M3 - Article
C2 - 14871894
AN - SCOPUS:2342425404
SN - 0021-9258
VL - 279
SP - 16912
EP - 16917
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 17
ER -