Pol η–dependent DNA synthesis at stalled replication forks during S phase suppresses chronic fragile site instability by preventing checkpoint-blind under-replicated DNA in mitosis.

DNA polymerase delta from calf thymus has been purified to apparent homogeneity by a new procedure which utilizes hydrophobic interaction chromatography with phenyl-Sepharose at an early step to separate most of the calcium-dependent protease activity from DNA polymerase delta and alpha. The purified enzyme migrates as a single protein band on polyacrylamide gel electrophoresis under nondenaturing conditions. The sedimentation coefficient of the enzyme is 7.9 S, and the Stokes radius is 53 A. A molecular weight of 173K has been calculated for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the homogeneous enzyme reveals two polypeptides of 125 and 48 kDa. This subunit structure differs from that of DNA polymerase delta prepared by our previous procedure, which was composed of subunits of 60 and 49 kDa [Lee, M. Y. W. T., Tan, C.-K., Downey , K. M., & So, A. G. (1981) Prog . Nucleic Acid Res. Mol. Biol. 26, 83-96], suggesting that the 60-kDa polypeptide may have been derived from the 125-kDa polypeptide during enzyme purification, possibly as the result of cleavage of an unusually sensitive peptide bond. DNA polymerase delta is separated from DNA polymerase alpha by hydrophobic interaction chromatography on phenyl-Sepharose; DNA polymerase delta is eluted at pH 7.2 and DNA polymerase alpha at pH 8.5. DNA polymerase delta can also be separated from DNA polymerase alpha by chromatography on hydroxylapatite; DNA polymerase alpha binds to hydroxylapatite in the presence of 0.5 M KCl, whereas DNA polymerase delta is eluted at 90 mM KCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammalian DNA polymerase ␦ (pol ␦), a key enzyme of chromosomal DNA replication, consists of four subunits as follows: the catalytic subunit; p125, which is tightly associated with the p50 subunit; p68, a proliferating cell nuclear antigen (PCNA)-binding protein; and a fourth subunit, p12. In this study, the functional roles of the p12 subunit of pol ␦ were studied. The inter-subunit interactions of the p12 subunit were determined by yeast two-hybrid assays and by pulldown assays. These assays revealed that p12 interacts with p125 as well as p50. This dual interaction of p12 suggests that it may serve to stabilize the p125-p50 interaction. p12 was shown to be a novel PCNA-binding protein. This was confirmed by identification of a PCNA-binding motif at its N terminus by binding assays and by site-directed mutagenesis. The activities and reaction products of recombinant pol ␦ containing a p12 mutant defective in PCNA binding, as well as purified recombinant pol ␦ and its subassemblies, were analyzed. Our results indicate that p12 contributes to PCNA-dependent pol ␦ activity, i.e. the p12-PCNA interaction is functional. Our data indicate that both p12 and p68 are required for optimal pol ␦ activity. This supports the hypothesis that the interaction between pol ␦ and PCNA is a divalent one that involves p12 and p68. We propose a model in which pol ␦ interacts with PCNA via at least two of its subunits, and one in which p12 could play a role in stabilizing the overall pol ␦-PCNA complex as well as pol ␦ itself.Chromosomal DNA replication in eukaryotic cells requires the following three distinct DNA polymerases: polymerase ␣, polymerase ␦ (pol ␦), 3 and polymerase ⑀. DNA pol ␦ is the key enzyme that is thought to play a central role in the elongation of both the leading and the lagging strands of DNA and the maturation of Okazaki fragments (1-3). DNA pol ␦ was originally identified as a new type of DNA polymerase possessing an intrinsic 3Ј-5Ј-exonuclease activity (4). Mammalian pol ␦ holoenzyme consists of the p125 catalytic subunit (which harbors both 5Ј-3Ј DNA polymerase and 3Ј-5Ј-exonuclease activities) and a tightly associated second subunit p50; this core is associated with two other subunits, p68 and p12, that are also referred to as the third and fourth subunits (5-9). The function of pol ␦ as a chromosomal DNA polymerase is dependent on its association with PCNA, which functions as a molecular sliding clamp (10, 11). The third subunits of pol ␦ in both mammalian (p68/p66) and in yeast cells (Cdc27 in Schizosaccharomyces pombe and pol 32 in Saccharomyces cerevisiae) harbor a PCNA-binding motif, and it has been shown that this provides a PCNA interaction site for pol ␦ (12-16). However, the exact nature of the subunit contacts of mammalian pol ␦ with PCNA has yet to be clarified; we (17-20) and others (8) have reported that human pol ␦ p125 binds to PCNA, although other reports have come to the opposite conclusion (14, 21). There is also a report that the p50 subunit of mammalian pol ␦ binds to PCNA (21).The fou...

The yeast two-hybrid screening method was used to identify novel proteins that associate with human DNA polymerase ␦ (pol ␦). Two baits were used in this study. These were the large (p125) and small (p50) subunits of the core pol ␦ heterodimer. p50 was the only positive isolated with p125 as the bait. Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen. In this study, the interaction of PDIP38 with pol ␦ was further characterized. PDIP38 encodes a protein of 368 amino acids whose C terminus is conserved with the bacterial APAG protein and with the F box A protein. It was found that PDIP38 also interacts with proliferating cell nuclear antigen (PCNA). The ability of PDIP38 to interact with both the p50 subunit of pol ␦ and with PCNA was confirmed by pull-down assays using glutathione S-transferase (GST)-PDIP38 fusion proteins. The PCNA-PDIP38 interaction was also demonstrated by PCNA overlay experiments. The association of PDIP38 with pol ␦ was shown to occur in calf thymus tissue and mammalian cell extracts by GST-PDIP38 pull-down and coimmunoprecipitation experiments. PDIP38 was associated with pol ␦ isolated by immunoaffinity chromatography. The association of PDIP38 with pol ␦ could also be demonstrated by native gel electrophoresis.DNA replication is essential not only for duplication of the genome but also for maintenance of genomic integrity during DNA repair (1, 2). Chromosomal DNA replication in eukaryotic cells requires three distinct DNA polymerases-␣, -␦, and -⑀ (1-6). pol 1 ␦ is required for replication of the leading strand and for completion of the lagging strand synthesis at the replication fork (7). The action of pol ␦ as a processive enzyme requires its interaction with proliferating cell nuclear antigen (PCNA), which functions as a molecular sliding clamp (1). The core mammalian pol ␦ enzyme consists of a tightly associated heterodimer of 125-and 50-kDa subunits. pol ␦ has been shown recently (8 -14) to consist of at least four subunits, consisting of the core enzyme and two additional subunits in both yeast and mammalian systems. In the yeast Schizosaccharomyces pombe, Cdc27 and Cdm1 have been identified as the third and the fourth pol ␦ subunits, respectively, (8, 9). In Saccharomyces cerevisiae Pol32p has been identified as the homologue of the S. pombe third subunit (10). A human homologue of Cdc27, the KIAA0039 gene product (11-13), and p12, a human homologue of Cdm1 (14), have recently been identified and can be considered to be the third and fourth subunits of human pol ␦. Our laboratory has been interested in the identification of additional protein components that are involved in the formation of the pol ␦ replication complex. In this study we report the identification of two novel proteins, PDIP38 and PDIP46, that interact with the p50 subunit of pol ␦ by the use of the yeast two-hybrid (15, 16) screening method. PDIP38 was shown to be a PCNA-binding protein, and its interaction with pol ␦ was established by additional experiments. EXPERIMENTAL PR...

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