Identification and Characterization of Human DNA Polymerase β2, a DNA Polymerase β-Related Enzyme*

The BRCA1 COOH terminus (BRCT) motif is present in many nuclear proteins that contribute to cell cycle regulation or DNA repair. Polymerase chain reaction-based screening with degenerate primers targeted to the BRCT motif resulted in the isolation of a human cDNA for a previously unidentified DNA polymerase (designated DNA polymerase β2) that is closely related to DNA polymerase β (Pol β). The predicted Pol β2 protein contains a BRCT motif in its NH2-terminal region; its COOH-terminal region exhibits 33% sequence identity to a corresponding region of human Pol β. The Pol β2 gene is expressed in a tissue-specific manner, with transcripts being most abundant in testis. A fusion construct comprising Pol β2 and green fluorescent protein exhibited a predominantly nuclear localization in transfected HeLa cells. Recombinant human Pol β2 from insect cells exhibited substantial DNA polymerase activity, but it did not possess terminal deoxyribonucleotidyl transferase activity. A truncated Pol β2 mutant lacking the BRCT motif retained substantial DNA polymerase activity, whereas a mutant Pol β2 with two alanine point mutations within the DNA polymerase active site did not. These results indicate that Pol β2 is a Pol β-related DNA polymerase with a BRCT motif that is dispensable for its polymerase activity.

The human BRCA1 gene, which determines susceptibility to familial breast and ovarian cancer (1), encodes a predicted protein of 1863 amino acids whose NH 2 -terminal region contains a single RING finger, a domain present in various proteins that exhibit transactivation activity at the promoter of certain viral and cellular genes (1,2). However, the observations that individuals who inherit a mutant BRCA1 gene containing a stop codon at codon 1853 develop early onset breast cancer (3) and that COOH-terminal truncation of the BRCA1 protein impairs its ability to inhibit the growth of breast cancer cells (4), suggest that the COOH-terminal portion of BRCA1 is essential for the normal function of this protein.
Computer analysis has identified a conserved domain within the COOH-terminal region of BRCA1 that contains a repeated motif, BRCA1 COOH terminus (BRCT) 1 (5,6). The BRCT motif comprises ϳ95 amino acids and occurs as a tandem repeat in the COOH-terminal region of various proteins; it has also been detected as a tandem repeat in the NH 2 -terminal region or as a single copy in some proteins. The BRCT motif is widespread from bacteria to mammals in nuclear proteins that are important in regulation of the cell cycle or in DNA repair, including p53BP1, RAD9, XRCC1, RAD4, Ect2, REV1, Crb2, RAP1, terminal deoxyribonucleotidyl transferase (TdT), and three eukaryotic DNA ligases (5,6). Despite the functional diversity of these nuclear proteins, participation in checkpoints responsive to DNA damage appears to be a unifying theme. X-ray crystallographic analysis of the BRCT motif has suggested that it might mediate interaction between proteins that contain it (7).
DNA polymerase ␤ (Pol ␤) plays an important role in base excision repair in mammals (8 -11). The 39-kDa vertebrate protein is organized into a 31-kDa COOH-terminal domain that includes the polymerase active site (12) and an 8-kDa NH 2 -terminal domain that participates in binding to DNA and exhibits 5Ј-deoxyribose phosphodiesterase (lyase) activity (13). The presence of both polymerase and lyase activities suggests that Pol ␤ functions in "short-patch" base excision repair by catalyzing both the removal of a 5Ј-deoxyribose phosphate intermediate and the subsequent filling of the resultant singlenucleotide gap (14, 15). Pol ␤ is also implicated in "long-patch" base excision repair (14), suggesting functions in meiosis (16) and nucleotide excision repair (17,18). However, Pol ␤-deficient cells are sensitive to DNA-alkylating agents such as methylmethane sulfonate but not to other DNA-damaging agents such as ultraviolet radiation (19), suggesting that another unidentified Pol ␤-like DNA polymerase might function in "long-patch" repair (including nucleotide excision repair).
We have now isolated a human cDNA that encodes a Pol ␤-like protein, designated DNA polymerase ␤2 (Pol ␤2). The predicted Pol ␤2 protein contains a BRCT motif in its NH 2 -terminal * This work was supported in part by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan (to M. N.), by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science (to M. N.), and by a health science research grant for research on the human genome and gene therapy from the Ministry of Health and Welfare of Japan (H10-genome-001 to K. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM   region, and we describe its characterization as a Pol ␤-related DNA polymerase.

Cloning of Human Pol ␤2 cDNA-Degenerate primers, 5Ј-GTIGTI(T/ C)TNACNAA(T/C)AT(A/T/C)GG and 5Ј-CG(A/G/T)AT(A/G/T)AT(A/G)T-GNGTNAC(A/G)TC
, were designed on the basis of the sequence homology shared by the BRCT motifs of Schizosaccharomyces pombe cut5 and human XRCC1 and were used to screen cDNAs from human MDAH041 cells with the use of the polymerase chain reaction (PCR). Sequence analysis of a resulting PCR product revealed an incomplete open reading frame that encoded an amino acid sequence homologous to the BRCT domains of S. pombe cut5 and human XRCC1. Additional 5Ј sequences of this cDNA were obtained by 5Ј rapid amplification of cDNA ends, and 3Ј sequences were obtained by screening a human fetal brain cDNA library (Stratagene).
Fluorescence in Situ Hybridization-The chromosomal location of the human Pol ␤2 gene was determined by fluorescence in situ hybridization as described previously (20). A biotinylated human Pol ␤2 cDNA probe that specifies nucleotide positions was allowed to hybridize overnight at 37°C with R-banded chromosomes prepared from phytohemagglutinin-stimulated lymphocytes of normal donors. The slides were washed first for 10 min at 37°C in 2ϫ standard saline citrate containing 50% formamide and then for 15 min at room temperature in 1ϫ standard saline citrate. Hybridization signals were detected with the use of rabbit antibodies to biotin (Enzo) and fluorescein isothiocyanatelabeled goat antibodies to rabbit immunoglobulin G (Enzo). The chromosomes were counterstained with propidium iodide.
Northern Blot Analysis-Polyadenylated RNA isolated from the indicated human tissues (CLONTECH) was subjected to Northern blot analysis. Hybridization was performed in 50% formamide at 42°C for 24 h with a 32 P-labeled probe corresponding to the NH 2 -terminal region of human Pol ␤2 cDNA (1-720).

RESULTS AND DISCUSSION
Cloning of Human Pol ␤2 cDNA-To isolate human cDNAs for previously unidentified proteins that contain a BRCT motif, we performed PCR with degenerate primers based on the se- quence homology apparent between the BRCT motifs of S. pombe cut5, also known as rad4 (22), and human XRCC1 (23). A 114-base pair PCR product was then used to isolate a corresponding full-length human cDNA, the nucleotide sequence of which encodes a protein of 575 amino acids with a calculated molecular mass of 63,421 Da (Fig. 1A). The predicted protein, which we have termed Pol ␤2, contains a BRCT motif in its NH 2 -terminal region (Fig. 1B). The COOH-terminal portion of the predicted protein exhibits 33 and 27% sequence identity to corresponding regions of human Pol ␤ and human TdT, respectively (Fig. 1C). Tissue Distribution of Pol ␤2 mRNA-The distribution of Pol ␤2 mRNA among various normal human tissues was examined by Northern blot analysis. The Pol ␤2-specific probe recognized a 2.0-kilobase mRNA that was most abundant in testis and ovary and was present in smaller amounts in prostate, skeletal muscle, and pancreas (Fig. 2). The expression of Pol ␤2 in the testis suggests that, like Pol ␤, this enzyme may contribute to meiotic cell division during spermatogenesis. In addition, Northern blot analysis revealed the cell cycle-dependent expression of Pol ␤2 mRNA with higher expression being observed in quiescent and S-to M-phase cells (data not shown).
Subcellular Localization of Pol ␤2-To determine the subcellular localization of Pol ␤2, we transfected HeLa cells with an expression vector for a fusion construct comprising Pol ␤2 and green fluorescent protein (GFP) and then examined the cells by ultraviolet microscopy. The Pol ␤2-GFP fusion protein was localized predominantly to the nucleus, whereas GFP alone was present in both the cytoplasm and nucleus (Fig. 3A). The nuclear localization of Pol ␤2 is thus consistent with the presence of a putative nuclear localization signal in the COOHterminal region of the protein (Fig. 1A), as well as with its predicted function as a DNA polymerase.
Chromosomal Localization of the Human Pol ␤2 Gene-Mutations in the Pol ␤ gene that result in the loss of base excision repair have been frequently associated with certain types of cancer, including prostate (24), colon (25), bladder (26), kidney (27), lung (28), and stomach (29) tumors. To determine whether the human chromosomal region that contains the Pol ␤2 gene might also be associated with cancer, we determined the chromosomal localization of this gene. Fluorescence in situ hybridization analysis revealed that the Pol ␤2 gene is localized to human chromosome 10q24.3-25.1 (Fig. 3B). Loss of heterozygosity at this region has been associated with several cancers, including glioma (30), as well as lung (31), prostate (32), and bladder (33) tumors, suggesting that mutation or deletion of the Pol ␤2 gene may contribute to oncogenesis.
DNA Polymerase Activity of Recombinant Pol ␤2 and Its Regulation by the BRCT Motif-To determine whether Pol ␤2 actually possesses DNA polymerase or TdT activities, we first generated recombinant Pol ␤2 proteins, tagged at their COOH termini with Myc and His 6 epitope tags, in insect cells. Lysates of Sf9 cells expressing Pol ␤2-Myc-His 6 proteins were subjected to immunoblot analysis with antibodies specific for the Myc or His 6 tags. The tagged wild-type protein and a mutant protein (⌬Pol ␤2; amino acids 240 -575) lacking the BRCT motif yielded immunoreactive bands corresponding to the expected sizes of ϳ68 and 48 kDa, respectively, with both types of antibodies (Fig. 4). The recombinant proteins were then immunoprecipitated with antibodies to the Myc tag and assayed for DNA polymerase activity with activated DNA or poly(rA) as templates. The activity of Pol ␤ expressed in and purified from Escherichia coli as described previously (34) was also assayed. The specific activity of the recombinant enzymes was calculated from the measured activity and the amount of protein as determined by SDS polyacrylamide gel electrophoresis and densitometric scanning of the stained bands. The specific activity of Pol ␤ was thus estimated as 2030 units/nmol with activated DNA and 57,800 units/nmol with poly(rA) ( Table I). Pol ␤ did not possess TdT activity. Wild-type Pol ␤2 also exhibited DNA polymerase activity, although its specific activity was less than that of Pol ␤ (60 units/nmol with activated DNA and 2480 units/nmol with poly(rA)). Like Pol ␤, Pol ␤2 did not possess TdT activity. As a control, a Pol ␤2 mutant (D427A-D429A) in which conserved aspartate residues (35) were replaced by alanine showed neither DNA polymerase nor TdT activities. In contrast, the ⌬Pol ␤2 mutant that lacks BRCT motif retained substantial DNA polymerase activity (370 units/ nmol with activated DNA and 7810 units/nmol with poly(rA)), indicating that the BRCT domain is dispensable for DNA polymerase activity.
In summary, our results indicate that Pol ␤2 possesses DNA polymerase activity similar to that of Pol ␤. Given that the BRCT motif is thought to mediate protein-protein interaction, it is possible that interaction of Pol ␤2 with an unidentified protein (or proteins), such as DNA ligase, through the BRCT motif may regulate its DNA polymerase activity. The identification of Pol ␤2 also raises the possibility that Pol ␤ and Pol ␤2 may cooperatively regulate base excision repair or nucleotide excision repair.