Proliferating Cell Nuclear Antigen Is Protected from Degradation by Forming a Complex with MutT Homolog2

Abstract

Proliferating cell nuclear antigen (PCNA) has been demonstrated to interact with multiple proteins involved in several metabolic pathways such as DNA replication and repair. However, there have been fewer reports about whether these PCNAbinding proteins influence stability of PCNA. Here, we observed a physical interaction between PCNA and MutT homolog2 (MTH2), a new member of the MutT-related proteins that hydrolyzes 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP). In several unstressed human cancer cell lines and in normal human fibroblast cells, PCNA and MTH2 formed a complex and their mutual binding fragments were confirmed. It was intriguing that PCNA and MTH2 were dissociated dependent on acetylation of PCNA, which in turn induced degradation of PCNA in response to UV irradiation, but not in response to other forms of DNA-damaging stress. To further explore the link between dissociation of PCNA-MTH2 and degradation of PCNA, RNAi against MTH2 was performed to mimic the dissociated status of PCNA to evaluate changes in the half-life of PCNA. Knockdown of MTH2 significantly promoted degradation of PCNA, suggesting that the physiological interaction of PCNA-MTH2 may confer protection from degradation for PCNA, whereas UV irradiation accelerates PCNA degradation by inducing dissociation of PCNA-MTH2. Moreover, secondary to degradation of PCNA, UV-induced inhibition of DNA synthesis or cell cycle progression was enhanced. Collectively, our data demonstrate for the first time that PCNA is protected by this newly identified partner molecule MTH2, which is related to DNA synthesis and cell cycle progression.

CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis

“…Since PCNA acetylation on Lys14 was suggested to modulate its degradation after UV damage (33), we reasoned that if a correlation exists between PCNA acetylation and its removal from chromatin for degradation, this may be dependent on CBP/p300 activity. To test this hypothesis, LF-1 fibroblasts were incubated in the presence of not-targeting, or CBP/p300 siRNA to deplete their protein levels, locally UV-irradiated and collected after 30 min or 4 h. No significant effect on the recruitment of PCNA to DNA damage sites was found at 30 min after UV (Supplementary Figure S5A), while a retention of PCNA was observed after 4 h in CBP/p300 depleted fibroblasts, as compared with fibroblasts incubated with non-targeting siRNA (Figure 6A).…”

“…Acetylation of PCNA was previously reported in proliferating cells, and after UV damage (31,33); however, its role in DNA repair has remained unknown. In addition, the molecular mechanism responsible for PCNA acetylation was not investigated, except for the interaction with p300 (36).…”

“…Acetylated lysines (Lys77, 80 and 248) were identified by mass spectrometry (MS) coupled to stable isotope labeling by amino acids in culture (SILAC) of mammalian cells (32). Mutational studies indicated that PCNA acetylation at Lys14 promoted its degradation after ultraviolet (UV) damage to inhibit DNA replication (33). However, the mechanism controlling PCNA removal from chromatin and its degradation after UV-induced nucleotide excision repair is unknown.…”

Multiple Nudix family proteins possess mRNA decapping activity

“…Expression of mouse Nudt15 in E. coli mutT − cells significantly reduced the elevated level of spontaneous mutation frequency, suggesting that it may function to minimize deleterious nucleotide incorporation into DNA (Cai et al 2003). Nudt15 is also implicated in DNA replication and repair by directly interacting with and stabilizing the PCNA protein (Yu et al 2009). Intriguingly, Nudt1 and Nudt5 are also 8-OH-dGTPases; however, they do not have RNA decapping activity ( Fig.…”

Suppression of mutagenesis by 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (7,8-dihydro-8-oxo-2′-deoxyguanosine 5′-triphosphate) by human MTH1, MTH2, and NUDT5

“…Recently, a physical interaction between MTH2 and PCNA was reported [41]. The knock-down of MTH2 significantly promoted the degradation of PCNA, and UV irradiation accelerated PCNA degradation by inducing the dissociation of PCNA-MTH2.…”