Our previous experiments showed a different behavior between pol d heterotetramer and trimer lacking p12 to WY 14643 citations perform translesion synthesis on templates containing base lesions . In order to reveal the role of pol d involved in BER, we examined cell extract and purified protein mediated uracil-intiated BER in vitro with uracilcontaining plasmid DNA. As shown in Figure 8B, left panel, the pol b 2/2 cell extract did not show a strong BER deficiency under the conditions in this assay, compared with extract from the wild-type MEF cell line . Pol b 2/2 cell extract still retained significant HhAntag691 repair activity, suggesting the presence of a pol b-independent BER back-up pathway. We depleted pol d four-subunit complex from wild type MEF and pol b 2/2 cell extract by using the antibodies against its individual four subunits . As expected, the level of BER significantly decreased, but a substantial level of BER still persisted in pol d�Cdepleted pol b 2/2 cell extract , which implies that pol d plays a significant role in BER. On the other hand, there may also exist other possible pol b-independent BER pathways, for example, pol l or pol q. To understand how pol d plays a role in BER, we monitored the ratio of two BER subpathways in reconstitution BER assay with purified recombinant proteins by measuring the SN BER and LP BER repair patch size using uracil-containing plasmid pUC19N as described in Materials and Methods. In such case, the kpnI-XhoI digested repair products resulted in a 32P labeled 25-bp fragment in which the uracil was replaced by dCMP at the first C, representing SN BER plus LP BER, whereas incorporation of dCMP at the second C and beyond resulted in a 32P labeled 16-bp fragment, representing LP BER . The amount of the SN BER product was calculated by subtracting the radioactivity of the 16-bp fragment from that of the 25-bp fragment. As shown in Figure 8C and 8D, pol d4 is more likely to mediate LP BER , compared with 42% of SN BER, while pol d3-p12 is more likely to mediate SN BER , compared with 34% of LP BER. Thus, it seems likely that loss of p12 modulates the rate of SN BER and LP BER during the repair process. These findings provide novel insights into the role of p12 in human pol d function and into the potential cellular consequences of the in vivo conversion of pol d4 to pol d3-p12 .