Human DNA-(Cytosine-5) Methyltransferase-PCNA Complex as a Target for p21 ^WAF1

Abstract

DNA-(cytosine-5) methyltransferase (MCMT) methylates newly replicated mammalian DNA, but the factors regulating this activity are unknown. Here, MCMT is shown to bind proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA replication and repair. Binding of PCNA requires amino acids 163 to 174 of MCMT, occurs in intact cells at foci of newly replicated DNA, and does not alter MCMT activity. A peptide derived from the cell cycle regulator p21(WAF1) can disrupt the MCMT-PCNA interaction, which suggests that p21(WAF1) may regulate methylation by blocking access of MCMT to PCNA. MCMT and p21(WAF1) may be linked in a regulatory pathway, because the extents of their expression are inversely related in both SV40-transformed and nontransformed cells.

Global delay in nascent strand DNA methylation

“…DNMT1 is also recruited to nascent DNA by the essential cofactor UHRF1 (ubiquitin-like, with PHD and RING finger domains 1), which exhibits a high affinity for hemi-methylated DNA through its SRA domain 8,9 and ubiquitinates the histone H3 tail to facilitate DNMT1 recruitment 10 . DNMT1 activity is further directed to the replication fork through its interaction with the proliferating cell nuclear antigen (PCNA) DNA clamp 11 , and deletion of DNMT1s PCNA-binding domain has been reported to delay post replication remethylation 12 . More conceptually, accurate reestablishment of the human methylome requires catalytic activity at ~45 million heterogeneously distributed CpGs (roughly 80% of CpG sites within the diploid genome) that must be completed within a single cell cycle 13 .…”

Expression of DNA methyl-transferase (DMT) and the cell cycle in human breast cancer cells

“…The cell cycle inhibitor p21 CIP1 can disrupt the association between DMT and PCNA, perhaps a ecting the activity of the two proteins (Chuang et al, 1997). Furthermore, levels of DMT and p21 proteins were inversely related in normal and SV-40 transformed human ®broblasts.…”

“…In contrast, PCNA expression did not vary greatly among the cell lines, and was not signi®cantly correlated with DMT expression. It has been suggested (Chuang et al, 1997) that the transforming e ect of DMT overexpression observed by others (Wu et al, 1993) may be due in part to the ability of DMT to compete with p21 for PCNA binding, thereby promoting the G 1 -S phase transition. By binding to PCNA in place of p21, excess DMT could increase the level of active cyclin-dependent kinases, promoting Rb phosphorylation and thus progression through the cell cycle.…”

“…A recent study reported that DMT can bind to PCNA (proliferating cell nuclear antigen), and that p21 CIP1 can disrupt the association between DMT and PCNA, possibly a ecting the activity of these proteins (Chuang et al, 1997). We therefore examined the levels of PCNA and p21 in the panel of ER-positive andnegative breast cancer cells (Figure 1).…”

Ras induces p21Cip1/Waf1 cyclin kinase inhibitor transcriptionally through Sp1-binding sites

“…p21 has several cellular binding partners, including GADD45 damage response protein (Kearsey et al, 1995) and PCNA (Chen et al, 1995). The sequestration of PCNA by p21 from the replication machinery proteins inhibits DNA replication (Flores-Rozas et al, 1994) and interferes with the interaction of PCNA with DNA methyltransferases (Chuang et al, 1997). The loss of p21 leads to defective damage responses and loss of G 1 and mitotic checkpoint control (Brugarolas et al, 1995;Deng et al, 1995).…”