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The gene for the mammalian DNA repair enzyme DNA polymerase β (fiβ-pol) is constitutively expressed in most cells, but is regulated in a tissue-specific fashion and can be induced in response to some types of DNA damaging agents. The promoter for the human β-pol gene has been characterized and found to be TATA-less, but it does have multiple GC boxes and one ATF/CRE-bindlng site located within 50 residues 5′ of the major mRNA start site. The ATF/CRE-bindlng site has been found to be essential for activity of the cloned promoter. We report that a bovine testes DNA-binding protein with specificity for the β-pol promoter ATF/CRE-binding site is phosphorylated in vivo and contains several phosphorylation sites. Sequence specific DNA-binding by the purified protein is reduced when the natural protein is dephosphorylated or when it is hyperphosphorylated by protein kinase A (cKA) in vitro. These results suggest the possibility that phosphorylation systems may change binding of this ATF/CRE-blnding protein to the β-pol promoter and in turn modulate the promoter. Possible correlation of the results with transient expression activity of the cloned β-pol promoter fusion gene was obtained in 293 cells. Cotransfection with a cKA expression plasmid to elevate phosphorylation was found to strongly reduce promoter activity.

DNA polymerase β (β-pol) is a single-copy gene that is considered to be part of the DNA repair machinery in mammalian cells. Using two human genomic libraries we have cloned the complete human β-pol gene and determined the organization of the β-pol coding sequence within the gene. The human β-pol gene spans 33 kb and contains 14 exons that range from 50 to 233 bp. The 13 introns vary from 96 bp to 6.5 kb. Information derived from this study was used in defining the location of a deletion/insertion type restriction fragment length polymorphism (RFLP) 5′ to exon I of the human β-pol gene. This RFLP was utilized in linkage analysis of DNAs from CEPH families and the results confirm the previous assignment of the human β-pol gene to chromosome 8 (p12-p11). Analysis of mRNA from six human cell lines using the polymerase chain reaction showed the expression of two β-pol transcripts. Sequence analysis revealed that the size difference in these transcripts was due to deletion of the 58 bp sequence encoded by exon II, suggesting that the smaller transcript results from an alternative splicing of the exon II sequence during processing of the β-pol precursor RNA.

We have shown by activity gel that overexpression in E.coli of a yeast chromosome 3 open reading frame (ORF) designated YCR14C and bearing homology to mammalian DNA polymeraseβ results in a new DNA polymerase in the host cells. The molecular mass of this enzyme corresponded to the YCR14C-predicted 67 kDa protein, and NH2-terminal amino acid sequencing confirmed that the expressed protein was encoded by the yeast ORF. This new yeast DNA polymerase was purified to homogeneity from E.coli. In a fashion similar to that of mammalian β-polymerases, the purified yeast enzyme exhibited distributive DNA synthesis on DNA substrate with a single-stranded template and processive gap-filling synthesis on a short-gapped DNA substrate. Activity of this yeast β-polymerase-like enzyme was sensitive to the β-polymerase inhibitor ddNTP and resistant to both 1 mM NEM and neutralizing antibody to E.coli DNA polymerase I. These results, therefore, indicate that YCR14C encodes a DNA β- polymerase-like enzyme in yeast, and we name it DNA polymerase IV. Yeast strains harboring a deletion mutation of the pol IV gene are viable, they exhibit no increase in sensitivity to ultraviolet light, ionizing radiation or alkylating agents, and sporulation and spore viability are not affected in the mutant.