Cdk-interacting protein 1 (Cipl) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initlafly observed in complexes containing CDK4, cyclin D, and proliferating celi nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymermse (po1) 8, and these three proteins constitute the p01 6 holoenzyme. Cipl is regulated by the tumor suppressor gene p53, a transcription factor that controls the G1 cell cycle arrest checkpoint in response to DNA damage (6). Cipl is transcriptionally induced by DNA damage in a p53-dependent fashion and may mediate cell cycle arrest (7). The G1 checkpoint is thought to arrest the cell cycle to prevent DNA replication of damaged templates while allowing repair.Although kinases are thought to regulate DNA synthesis, the appearance ofCipl and PCNA in a complex suggested the possibility of a more direct link to replication. PCNA, in conjunction with activator 1 (Al; also known as RF-C), acts as a processivity factor for DNA polymerase (pol) 8 (8-10).Recent studies have shown that PCNA (as a trimer) and the f3 subunit (a dimer) of DNA pol III holoenzyme of Escherichia coli both form a tight clamp that tethers their cognate DNA polymerase to the template and translocates along the DNA with the polymerase during replication (11)(12)(13). Thus any agent that can interact with the sliding clamp might impede the movement of the replication complex.Here we demonstrate that Cipl directly binds PCNA to form a complex that inhibits the activity of the PCNAdependent pol 8 holoenzyme, which reduces the replication of simian virus 40 (SV40) DNA and the elongation of primed DNA templates. However, SV40 DNA replication carried out with the pol 8-independent monopolymerase system was unaffected by Cipl. The inhibitory effects of Cipl were independent of the cdk2-cyclin A complex. MATERIALS AND METHODSPreparation of DNAs and Proteins. (dT)1s18 and (dA)4500 were obtained from Life Sciences Inc.; poly(dA) was annealed to oligo(dT) (20:1) as described (14). Singly primed DNA was prepared by hybridizing a 2-fold molar excess of a 34-nt oligonucleotide to circular single-stranded M13 mp7 (7.2 kb) DNA (containing 10-15% linear molecules) at nucleotide positions 6300-6633. The annealed product was labeled by the incorporation of a single dCMP residue (corresponding to position 6299) using the Klenow fragment of DNA pol I. The resulting product (=2000 cpm/fmol), after phenol extraction, was isolated by filtration through a G-50

The Saccharomyces cerevisiae genome encodes four MutL homologs. Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches. We have investigated the role of MLH3 in mismatch repair. Mutations in MLH3 increased the rate of reversion of the hom3-10 allele by increasing the rate of deletion of a single T in a run of 7 Ts. Combination of mutations in MLH3 and MSH6 caused a synergistic increase in the hom3-10 reversion rate, whereas the hom3-10 reversion rate in an mlh3 msh3 double mutant was the same as in the respective single mutants. Similar results were observed when the accumulation of mutations at frameshift hot spots in the LYS2 gene was analyzed, although mutation of MLH3 did not cause the same extent of affect at every LYS2 frameshift hot spot. MLH3 interacted with MLH1 in a two-hybrid system. These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex.

Proliferating cell nuclear antigen (PCNA) is required for mismatch repair (MMR) and has been shown to interact with complexes containing Msh2p or MLH1 (refs 1-4). PCNA has been implicated to act in MMR before and during the DNA synthesis step, although the biochemical basis for the role of PCNA early in MMR is unclear. Here we observe an interaction between PCNA and Msh2p-Msh6p mediated by a specific PCNA-binding site present in Msh6p. An msh6 mutation that eliminated the PCNA-binding site caused a mutator phenotype and a defect in the interaction with PCNA. The association of PCNA with Msh2p-Msh6p stimulated the preferential binding of Msh2p-Msh6p to DNA containing mispaired bases. Mutant PCNA proteins encoded by MMR-defective pol30 alleles were defective for interaction with Msh2p-Msh6p and for stimulation of mispair binding by Msh2p-Msh6p. Our results suggest that PCNA functions directly in mispair recognition and that mispair recognition requires a higher-order complex containing proteins in addition to Msh2p-Msh6p.