Enzymic Research On Nucleotide Excision Repair Related Protein XPB Bax1in Pyrococcus Furiosus

Pyrococcus furiosus belongs to heat resistant archaea. Nucleotideexcision repair is a DNA repair mechanism that removes bulky DNAadducts resulting from UV induced DNA damage. In order to do researchon NER related protein XPB and Bax1, several pyrococcus furiosusgenes including pfuXPB, pfuBax1, pfuPCNA, pfuRPA, etc. were clonedby vector pDest17. All the proteins were successfully expressed. Afterseveral purification process, including Ni-NTA chromatography, ionexchange chromatography and gel filtration, we obtained homogeneousproteins. Then we studied the enzymic activities of these proteins in vitroand discussed the possible mechanism of how XPB and Bax1participatein NER. We obtained following results.pfuXPB is homologous to human XPB. It can binds to DNAsubstrates. It also has ATPase activity. Meantime, it can binds to pfuBax1 to form heterodimer. After pfuXPB and pfuBax1combine with each other,their ability to binds to DNA will be greatly enhanced and the ATPaseactivity of pfuXPB will also be strengthened. However, as homologoushuman XPB, we did not observe the expected helicase activity of pfuXPB.pfuBax1is a protein specific to archaea and it belongs to the same operonwith pfuXPB. The two proteins were proved to have physical interactionsand can affect each other. PfuXPB can trigger pfuBax1’s nuclease activity,making pfuBax1excise DNA bubble substrates. In order to excise DNAbubble substrates, XPB/Bax1complex needs the existence of divalentmetal ion. The convertion of divalent metal ion will change the excisionpattern of XPB/Bax1complex. For example, in this paper, we found thatXPB/Bax1complex had different excision patterns between Mg2+andMn2+conditions. The complex can only excise DNA bubble substrates inMg2+condition while it can also excise single strand DNA substrates withMn2+.In order to further study the XPB/Bax1complex’s excision pattern,we utilized various DNA structure as substrates to test the excision ability.Through analysis, we made our own hypothesis on how XPB/Bax1complex carried out excision process.Meanwhile, we added pfuPCNA, pfuRPA and histone to the excisionbuffer to test their influence to XPB/Bax1complex’s nuclease activity. Itturned out that pfuRPA and pfuhistone had little effects on the excision pattern while pfuPCNA can make a big difference on the complexexcision pattern. Through further analysis and mutation experiments, wedemonstrated our hypothesis.