Effec of MMS on protein interaction with human MPG

In mammalian cells, DNA constantly suffers from lesions such as strand breaks and damaged bases. Most damaged bases are repaired by base excision repair (BER) pathways. Human N-methylpurine DNA glycosylase (hMPG) excises various types of damaged purines generated by alkylation or deamination, and it is known to move over DNA and detect damaged bases. The abundance of hMPG in human cell is estimated to be about 2 x 10⁵ molecules per cell. On the other hand, it is estimated that about 1 x 10⁴ altered bases are formed in human genome every day. Thus it is possible that there is a very efficient system of damage sensing for rare lesions. hMPG interacts with various proteins including some transcriptional regulators, but it remains unclear what factors are associated with hMPG damage sensing. In this study, we performed pull down assay with purified GST-hMPG and nuclear extract from MMS-treated HeLa cells to identify the protein interacted with hMPG. The result showed that hMPG interacts with XRCC1, MBD1 and PCNA. The result was consistent with previous reports which suggested physical interactions of these proteins. Furthermore, by MMS treatment, the levels of XRCC1 and MBD1 in pull down fraction are increased 15.9 and 2.1-fold, respectively. To investigate the interactions in vivo, we prepared HeLa cell expressing GST-hMPG. The expression was confirmed by western blotting analysis with anti-hMPG and GST antibody. Now, we are investigating the effect of MMS on proteins interaction with hMPG through pull down assay.