Helicase activity and substrate specificity of RecQ5β
|
摘要
RecQ5β is an essential DNA helicase in humans, playing important roles in DNA replication, repair, recombination and transcription. The unwinding activity and substrate specificity of RecQ5β is still elusive. Here, we used stopped-flow kinetic method to measure the unwinding and dissociation kinetics of RecQ5β with several kinds of DNA substrates, and found that RecQ5β could well unwind ss/ds DNA, forked DNA and Holiday junction, but was compromised in unwinding blunt DNA and G-quadruplex. Rec5β has the preferred unwinding specificity for certain DNA substrates containing the junction point, which may improve the binding affinity and unwinding activity of RecQ5β. Moreover, from a comparison with the truncated RecQ5β~(1-467), we discovered that the C-terminal domain might strongly influence the unwinding activity and binding affinity of RecQ5β. These results may shed light on the physiological functions and working mechanisms of RecQ5β helicase.
RecQ5β is an essential DNA helicase in humans, playing important roles in DNA replication, repair, recombination and transcription. The unwinding activity and substrate specificity of RecQ5β is still elusive. Here, we used stopped-flow kinetic method to measure the unwinding and dissociation kinetics of RecQ5β with several kinds of DNA substrates, and found that RecQ5β could well unwind ss/ds DNA, forked DNA and Holiday junction, but was compromised in unwinding blunt DNA and G-quadruplex. Rec5β has the preferred unwinding specificity for certain DNA substrates containing the junction point, which may improve the binding affinity and unwinding activity of RecQ5β. Moreover, from a comparison with the truncated RecQ5β~(1-467), we discovered that the C-terminal domain might strongly influence the unwinding activity and binding affinity of RecQ5β. These results may shed light on the physiological functions and working mechanisms of RecQ5β helicase.
RecQ5β is an essential DNA helicase in humans, playing important roles in DNA replication, repair, recombination and transcription. The unwinding activity and substrate specificity of RecQ5β is still elusive. Here, we used stopped-flow kinetic method to measure the unwinding and dissociation kinetics of RecQ5β with several kinds of DNA substrates, and found that RecQ5β could well unwind ss/ds DNA, forked DNA and Holiday junction, but was compromised in unwinding blunt DNA and G-quadruplex. Rec5β has the preferred unwinding specificity for certain DNA substrates containing the junction point, which may improve the binding affinity and unwinding activity of RecQ5β. Moreover, from a comparison with the truncated RecQ5β~(1-467), we discovered that the C-terminal domain might strongly influence the unwinding activity and binding affinity of RecQ5β. These results may shed light on the physiological functions and working mechanisms of RecQ5β helicase.
引文
[1]Lohman T M and Bjornson K P 1996 Ann.Rev.Biochem.65 169
[2]Soultanas P and Wigley D B 2000 Curr.Opin.Struct.Biol.10 124
[3]Caruthers J M and Mckay D B 2002 Curr.Opin.Struct.Biol.12 123
[4]Umezu K,Nakayama K and Nakayama H 1990 Proc.Natl.Acad.Sci.87 5363
[5]Lohman T M,Tomko E J and Wu C G 2008 Nat.Rev.Mol.Cell Bio.9391
[6]Masai H 2011 J.Biochem.149 629
[7]Mankouri H W and Hickson I D 2004 Biochem.Soc.T.32 957
[8]Hanada K,Ukita T,Kohno Y,Saito K,Kato J and Ikeda H 1997 Proc.Natl.Acad.Sci.94 3860
[9]German J,Schonberg S,Louie E and Chaganti R S 1977 Am.J.Hum.Genet.29 248
[10]Speina E,Dawut L,Hedayati M,Wang Z,May A,Schwendener S,Janscak P,Croteau D L and Bohr V A 2010 Nucleic Acids Res.38 2904
[11]Seki M 1994 Nucleic Acids Res.22 4566
[12]Sharma S,Stumpo D J,Balajee A S,Bock C B,Lansdorp P M,Brosh R M Jr and Blackshear P J 2007 Mol.Cell.Biol.27 1784
[13]Croteau D L,Popuri V,Opresko P L and Bohr V A 2014 Ann.Rev.Biochem.83 519
[14]Karow J K,Wu L and Hickson I D 2000 Curr.Opin.Genet.Dev.10 32
[15]Wang W,Seki M,Narita Y,Nakagawa T,Yoshimura A,Otsuki M,Kawabe Y,Tada S,Yagi H and Ishii Y 2003 Mol.Cell.Biol.23 3527
[16]Hu Y,Lu X,Barnes E,Yan M,Lou H and Luo G 2005 Mol.Cell.Biol.25 3431
[17]Paliwal S,Kanagaraj R,Sturzenegger A,Burdova K and Janscak P2014 Nucleic Acids Res.42 2380
[18]Shimamoto A,Nishikawa K,Kitao S and Furuichi Y 2000 Nucleic Acids Res.28 1647
[19]Popuri V,Tadokoro T,Croteau D L and Bohr V A 2013 Crit.Rev.Biochem.Mol.Biol.48 289
[20]Sekelsky J J,Brodsky M H,Rubin G M and Hawley R S 1999 Nucleic Acids Res.27 3762
[21]Ding X Y,Xu Y N,Li W,Wang P Y,Xi X G and Dou S X 2012 Chin.Sci.Bull.57 1280
[22]Kanagaraj R,Saydam N,Garcia P L,Zheng L and Janscak P 2006 Nucleic Acids Res.34 5217
[23]Garcia P L,Liu Y,Jiricny J,West S C and Janscak P 2004 EMBO J.232882
[24]Ren H,Dou S X,Zhang X D,Wang P Y,Kanagaraj R,Liu J L,Janscak P,Hu J S and Xi X G 2008 Biochem.J.412 425
[25]Mohaghegh P 2001 Nucleic Acids Res.29 2843
[26]Ozsoy A Z,Ragonese H M and Matson S W 2003 Nucleic Acids Res.31 1554
[27]Zhang X D,Dou S X,Xie P,Wang P Y and Xi X G 2005 Acta Bioch.Bioph.Sin.37 593
[28]Zhang X D,Dou S X,Xie P,Hu J S,Wang P Y and Xi X G 2006 J.Biol.Chem.281 12655
[29]Yang Y,Dou S X,Ren H,Wang P Y,Zhang X D,Qian M,Pan B Y and Xi X G 2008 Nucleic Acids Res.36 1976
[30]Yang Y,Dou S X,Xu Y N,Bazeille N,Wang P Y,Rigolet P,Xu H Q and Xi X G 2010 Biochemistry 49 656
[31]Ding X Y,Xu Y N,Wang P Y,Dou S X and Xi X G 2012 Afr.J.Biotechnol.11 6795
[32]Xu Y N,Bazeille N,Ding X Y,Lu X M,Wang P Y,Bugnard E,Grondin V,Dou S X and Xi X G 2012 Nucleic Acids Res.40 9802
[33]Xu H Q,Zhang A H,Auclair C and Xi X G 2003 Nucleic Acids Res.31 e70
[34]Machwe A,Xiao L,Groden J,Matson S W and Orren D K 2005 J.Biol.Chem.280 23397
[35]Valenti A,Perugino G,Varriale A,D’Auria S,Rossi M and Ciaramella M 2010 J.Biol.Chem.285 36532
[36]Paeschke K,Mc Donald K R and Zakian V A 2010 FEBS Lett.584 3760
[37]Gray R D and Chaires J B 2008 Nucleic Acids Res.36 4191
[38]Gray R D,Trent J O and Chaires J B 2014 J.Mol.Biol.426 1629
[39]Budhathoki J B,Maleki P,Roy W A,Janscak P,Yodh J G and Balci H2016 Biophys.J.110 2585
[2]Soultanas P and Wigley D B 2000 Curr.Opin.Struct.Biol.10 124
[3]Caruthers J M and Mckay D B 2002 Curr.Opin.Struct.Biol.12 123
[4]Umezu K,Nakayama K and Nakayama H 1990 Proc.Natl.Acad.Sci.87 5363
[5]Lohman T M,Tomko E J and Wu C G 2008 Nat.Rev.Mol.Cell Bio.9391
[6]Masai H 2011 J.Biochem.149 629
[7]Mankouri H W and Hickson I D 2004 Biochem.Soc.T.32 957
[8]Hanada K,Ukita T,Kohno Y,Saito K,Kato J and Ikeda H 1997 Proc.Natl.Acad.Sci.94 3860
[9]German J,Schonberg S,Louie E and Chaganti R S 1977 Am.J.Hum.Genet.29 248
[10]Speina E,Dawut L,Hedayati M,Wang Z,May A,Schwendener S,Janscak P,Croteau D L and Bohr V A 2010 Nucleic Acids Res.38 2904
[11]Seki M 1994 Nucleic Acids Res.22 4566
[12]Sharma S,Stumpo D J,Balajee A S,Bock C B,Lansdorp P M,Brosh R M Jr and Blackshear P J 2007 Mol.Cell.Biol.27 1784
[13]Croteau D L,Popuri V,Opresko P L and Bohr V A 2014 Ann.Rev.Biochem.83 519
[14]Karow J K,Wu L and Hickson I D 2000 Curr.Opin.Genet.Dev.10 32
[15]Wang W,Seki M,Narita Y,Nakagawa T,Yoshimura A,Otsuki M,Kawabe Y,Tada S,Yagi H and Ishii Y 2003 Mol.Cell.Biol.23 3527
[16]Hu Y,Lu X,Barnes E,Yan M,Lou H and Luo G 2005 Mol.Cell.Biol.25 3431
[17]Paliwal S,Kanagaraj R,Sturzenegger A,Burdova K and Janscak P2014 Nucleic Acids Res.42 2380
[18]Shimamoto A,Nishikawa K,Kitao S and Furuichi Y 2000 Nucleic Acids Res.28 1647
[19]Popuri V,Tadokoro T,Croteau D L and Bohr V A 2013 Crit.Rev.Biochem.Mol.Biol.48 289
[20]Sekelsky J J,Brodsky M H,Rubin G M and Hawley R S 1999 Nucleic Acids Res.27 3762
[21]Ding X Y,Xu Y N,Li W,Wang P Y,Xi X G and Dou S X 2012 Chin.Sci.Bull.57 1280
[22]Kanagaraj R,Saydam N,Garcia P L,Zheng L and Janscak P 2006 Nucleic Acids Res.34 5217
[23]Garcia P L,Liu Y,Jiricny J,West S C and Janscak P 2004 EMBO J.232882
[24]Ren H,Dou S X,Zhang X D,Wang P Y,Kanagaraj R,Liu J L,Janscak P,Hu J S and Xi X G 2008 Biochem.J.412 425
[25]Mohaghegh P 2001 Nucleic Acids Res.29 2843
[26]Ozsoy A Z,Ragonese H M and Matson S W 2003 Nucleic Acids Res.31 1554
[27]Zhang X D,Dou S X,Xie P,Wang P Y and Xi X G 2005 Acta Bioch.Bioph.Sin.37 593
[28]Zhang X D,Dou S X,Xie P,Hu J S,Wang P Y and Xi X G 2006 J.Biol.Chem.281 12655
[29]Yang Y,Dou S X,Ren H,Wang P Y,Zhang X D,Qian M,Pan B Y and Xi X G 2008 Nucleic Acids Res.36 1976
[30]Yang Y,Dou S X,Xu Y N,Bazeille N,Wang P Y,Rigolet P,Xu H Q and Xi X G 2010 Biochemistry 49 656
[31]Ding X Y,Xu Y N,Wang P Y,Dou S X and Xi X G 2012 Afr.J.Biotechnol.11 6795
[32]Xu Y N,Bazeille N,Ding X Y,Lu X M,Wang P Y,Bugnard E,Grondin V,Dou S X and Xi X G 2012 Nucleic Acids Res.40 9802
[33]Xu H Q,Zhang A H,Auclair C and Xi X G 2003 Nucleic Acids Res.31 e70
[34]Machwe A,Xiao L,Groden J,Matson S W and Orren D K 2005 J.Biol.Chem.280 23397
[35]Valenti A,Perugino G,Varriale A,D’Auria S,Rossi M and Ciaramella M 2010 J.Biol.Chem.285 36532
[36]Paeschke K,Mc Donald K R and Zakian V A 2010 FEBS Lett.584 3760
[37]Gray R D and Chaires J B 2008 Nucleic Acids Res.36 4191
[38]Gray R D,Trent J O and Chaires J B 2014 J.Mol.Biol.426 1629
[39]Budhathoki J B,Maleki P,Roy W A,Janscak P,Yodh J G and Balci H2016 Biophys.J.110 2585