Abstract
The diverse function of proliferating cell nuclear antigen (PCNA) may be regulated by interactions with different protein partners. Interestingly, the binding sites for all known PCNA-associating proteins are on the outer surface or the C termini ("front") sides of the PCNA trimer. Using cell extracts and purified human PCNA protein, we show here that two PCNA homotrimers form a back-to-back doublet. Mutation analysis suggests that the Arg-5 and Lys-110 residues on the PCNA back side are the contact points of the two homotrimers in the doublet. Furthermore, short synthetic peptides encompassing either Arg-5 or Lys-110 inhibit double trimer formation. We also found that a PCNA double trimer, but not a homotrimer alone, can simultaneously accommodate chromatin assembly factor-1 and polymerase delta. Together, our data supports a model that chromatin remodeling by chromatin assembly factor-1 (and, possibly, many other cellular activities) are tightly coupled with DNA replication (and repair) through a PCNA double trimer complex.
Highlights
The connection between chromatin assembly factor-1 (CAF-1) and DNA replication was initially suggested to be that CAF-1 was found in the replication foci [17]
We found that a proliferating cell nuclear antigen (PCNA) double trimer, but not a homotrimer alone, can simultaneously accommodate chromatin assembly factor-1 and polymerase ␦
Our data supports a model that chromatin remodeling by chromatin assembly factor-1 are tightly coupled with DNA replication through a PCNA double trimer complex
Summary
The connection between CAF-1 and DNA replication was initially suggested to be that CAF-1 was found in the replication foci [17]. When cells were treated with 1.5% formaldehyde for 30 min to cross-link proteins prior to SDS-PAGE, PCNA was detected at the positions of 33, ϳ100, and ϳ200 kDa (Fig. 1A, lane 2). When the cross-linking time was extended to 90 min, essentially all of the PCNA molecules in the cells formed a ϳ200-kDa protein complex (Fig. 1A, lane 3).
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