| Abstract: | Chromatin assembly factor 1 (CAF-1) deposits histones H3 and H4 rapidly behind replication forks through an interaction with the proliferating cell nuclear antigen (PCNA), a DNA polymerase processivity factor that also binds to a number of replication enzymes and other proteins that act on nascent DNA. The mechanisms that enable CAF-1 and other PCNA-binding proteins to function harmoniously at the replication fork are poorly understood. Here we report that the large subunit of human CAF-1 (p150) contains two distinct PCNA interaction peptides (PIPs). The N-terminal PIP binds strongly to PCNA in vitro but, surprisingly, is dispensable for nucleosome assembly and only makes a modest contribution to targeting p150 to DNA replication foci in vivo. In contrast, the internal PIP (PIP2) lacks one of the highly conserved residues of canonical PIPs and binds weakly to PCNA. Surprisingly, PIP2 is essential for nucleosome assembly during DNA replication in vitro and plays a major role in targeting p150 to sites of DNA replication. Unlike canonical PIPs, such as that of p21, the two p150 PIPs are capable of preferentially inhibiting nucleosome assembly, rather than DNA synthesis, suggesting that intrinsic features of these peptides are part of the mechanism that enables CAF-1 to function behind replication forks without interfering with other PCNA-mediated processes.Eukaryotic cells in S phase not only have to replicate their entire genome but also faithfully reproduce preexisting chromatin structures onto the two nascent chromatids. The duplication of chromatin structures during DNA replication is a challenging task for eukaryotic cells. Newly synthesized histones are deposited very rapidly behind replication forks (150 to 300 bp), almost as soon as enough DNA has emerged from the replisome to allow the formation of nucleosome core particles (52). A key protein involved in coupling nucleosome assembly to DNA replication is chromatin assembly factor 1 (CAF-1). CAF-1 is a complex of three polypeptide subunits, known as p150, p60, and RbAp48 in vertebrates, that mediates the first step in nucleosome formation by depositing newly synthesized histone H3/H4 onto DNA (25, 50).In mouse and human cells, CAF-1 localizes to virtually all DNA replication foci throughout the S phase (28, 38, 49, 54). This strongly argues that CAF-1 is a physiologically relevant histone H3/H4 nucleosome assembly factor. In addition, disruption of CAF-1 function in human cells results in a severe loss of viability that is accompanied by spontaneous DNA damage and a block in S-phase progression (20, 40, 60). Thus, unlike in Saccharomyces cerevisiae, the function of CAF-1 in vertebrates cannot be replaced by that of other nucleosome factors, such as members of the Hir protein family or Rtt106 (24, 27, 29). This may be because, unlike CAF-1, HIRA (a human homologue of yeast Hir1 and Hir2) does not associate with core histones that are synthesized during S phase (55). In human cells, the ability to promote nucleosome assembly preferentially onto replicating DNA is thus far unique to CAF-1.This distinctive property of CAF-1 is mediated through proliferating cell nuclear antigen (PCNA), a homotrimeric ring that encircles double-stranded DNA (4) and acts as a sliding clamp to tether DNA polymerases to their DNA substrate and thereby enhance their processivity. Several lines of biochemical and genetic evidence support the role of PCNA in CAF-1-mediated nucleosome assembly. First, CAF-1 colocalizes with PCNA in vivo and binds directly to PCNA in vitro (27, 35, 49, 61). Second, even in the presence of excess unreplicated DNA, CAF-1 can select fully replicated plasmid DNA molecules as preferential substrates for histone deposition, but only when those molecules are associated with PCNA (49). Third, PCNA-driven DNA synthesis can also attract CAF-1 to sites of DNA repair events, such as nucleotide excision repair (12, 15, 32, 35). Fourth, a specific PCNA mutation impairs the role of CAF-1 in telomeric silencing in S. cerevisiae (48, 61). Interestingly, a number of PCNA mutations that reduce its interaction with other PCNA-binding proteins have apparently no effect on CAF-1 function in vivo (48, 61). This implies that the interaction of CAF-1 with PCNA is substantially different from that of other PCNA-binding proteins.Enhancing DNA polymerase processivity is not the only function of PCNA in DNA replication. The sliding clamp also directly binds to other replication enzymes, such as DNA ligase 1, DNA polymerase δ, and FEN1 (14, 21, 37). In addition to its roles in DNA synthesis and nucleosome assembly, PCNA also directly binds to a number of enzymes that continuously monitor and correct the quality of nascent DNA. These include enzymes involved in epigenetic inheritance, such as the maintenance DNA methyltransferase DNMT1 (8), base excision repair (UNG2) (42), mismatch repair (MSH3 and MSH6) (9), DNA lesion bypass (23), and many other processes (31, 36). Even subtle defects in many of these processes, including CAF-1-dependent nucleosome assembly (39), lead to either chromosome rearrangements or mutator phenotypes, which are common features of many human cancers. Surprisingly, many of these enzymes interact with PCNA via canonical PCNA interaction peptides (PIPs) that conform to the consensus sequence QXXhXXaa, where Q is a glutamine, h is a hydrophobic residue (valine, methionine, leucine, or isoleucine), a is an aromatic residue (phenylalanine, tyrosine, tryptophan, or occasionally histidine), and X represents any amino acid. Therefore, regulatory mechanisms must exist to ensure that these fundamentally distinct PCNA-dependent processes occur in a carefully orchestrated manner without mutually interfering with each other.In order to understand how the action of CAF-1 is coordinated with that of other PCNA-binding proteins at replication forks, we carried out a thorough study of CAF-1 PIPs by analyzing their functions using a number of assays. We found that the p150 subunit of CAF-1 contains two fundamentally distinct PIPs. The N-terminal motif (PIP1) binds strongly to PCNA in vitro but is dispensable for nucleosome assembly during simian virus 40 (SV40) DNA replication. In contrast, despite the lack of a key conserved residue, the second PIP (PIP2) of CAF-1 is crucial for replication-dependent nucleosome assembly in vitro and for targeting CAF-1 to DNA replication foci in vivo. Remarkably, although PIP2 exhibits some features of canonical PIPs, it binds only weakly to PCNA in vitro. We suggest that regulated PCNA binding via this peptide may play an important role in ensuring that CAF-1 can efficiently deposit histones behind replication forks without competing with the numerous other enzymes that require continuous access to PCNA during DNA replication. Consistent with this, we show that CAF-1 PIPs possess the ability to preferentially interfere with nucleosome assembly rather than with DNA synthesis. |
