Cloning and characterization of novel proteins which associate with mammalian DNA polymerase delta

The yeast two-hybrid screening method was used to identify and to clone novel proteins that associate with the DNA polymerase delta (pol delta)-PCNA complex. When the pol delta p125 subunit was used as the bait, the positive clones were all found to encode the pol delta p50 subunit. When PCNA was used as the bait, the positive clones were identified as p21waf1 and DNA (cytosine-5) methyltransferase, both of which are known to interact with PCNA. When p50 was used as a bait, three novel protein partners were identified. The first of these was p21waf1, which was previously unknown to interact with p50 This result was confirmed by p21-GST (glutathione S-transferase) fusion protein pull-down assays of pol delta p50 from both Hela and calf thymus cell extracts.The other two protein partners were found to be novel proteins of unknown function. These were named p42 and p46. p42 consists of 368 aa (amino acid residues) with a predicted molecular mass of 42 kDa, and a pI value of 8.91. It was also discovered that p42 interacts with PCNA. The ability of p42 to interact with both pol delta p50 and with PCNA was confirmed by additional yeast two-hybrid pairwise interaction assays, and by other independent methods.The cDNA sequence of p46 was determined and encodes a protein of 421 aa with a predicted molecular mass of 46 kDa and a pI value of 10.61. p46 interacted with pol delta p50 subunit but did not interact with pol delta p125 subunit or PCNA in the yeast two-hybrid assay.The gene and the open reading frame of the human homolog of S. pombe Cdm1 (hCdm1), the smallest subunit of pol delta, were identified by a database search. hCdm1 was composed of 107 aa with a predicted molecular mass of 12 kDa. p12/hCdm1 was shown to be associated with pol delta p125 and p50 subunits in both immunoaffinity purified pol delta and conventionally purified pol delta.These studies have identified three human proteins that interact with pol delta, and provide the foundation for further studies to determine their functions in DNA replication.