Nucleic Acids Research Advance Access originally published online on April 22, 2007
Nucleic Acids Research 2007 35(9):3053-3063; doi:10.1093/nar/gkm092
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2007, Vol. 35, No. 9 3053-3063
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Domain swapping between FEN-1 and XPG defines regions in XPG that mediate nucleotide excision repair activity and substrate specificity
1Institute of Molecular Cancer Research, University of Zurich, Switzerland, 2Department of Microbiology and Molecular Medicine, University Medical Centre, Geneva, Switzerland, 3Department of Pharmacological Sciences, SUNY Stony Brook, New York, USA, 4Department of Cell Biology and Genetics, Erasmus Medical Center, Rotterdam, The Netherlands
*To whom correspondence should be addressed. Tel: 631 632 7545; Fax: 631 632 7546; E-mail: orlando{at}pharm.stonybrook.edu
Received August 21, 2006. Revised February 1, 2007. Accepted February 2, 2007.
FEN-1 and XPG are members of the FEN-1 family of structure-specific nucleases, which share a conserved active site. FEN-1 plays a central role in DNA replication, whereas XPG is involved in nucleotide excision repair (NER). Both FEN-1 and XPG are active on flap structures, but only XPG cleaves bubble substrates. The spacer region of XPG is dispensable for nuclease activity on flap substrates but is required for NER activity and for efficient processing of bubble substrates. Here, we inserted the spacer region of XPG between the nuclease domains of FEN-1 to test whether this domain would be sufficient to confer XPG-like substrate specificity and NER activity on a related nuclease. The resulting FEN-1-XPG hybrid protein is active on flap and, albeit at low levels, on bubble substrates. Like FEN-1, the activity of FEN-1-XPG was stimulated by a double-flap substrate containing a 1-nt 3' flap, whereas XPG does not show this substrate preference. Although no NER activity was detected in vitro, the FEN-1-XPG hybrid displays substantial NER activity in vivo. Hence, insertion of the XPG spacer region into FEN-1 results in a hybrid protein with biochemical properties reminiscent of both nucleases, including partial NER activity.
Present addresses: Marcel Hohl, Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 10021 New York, USA; Isabelle Dunand-Sauthier, Department of Pathology and Immunology, University Medical Centre, Geneva, Switzerland; Fabrizio Thorel, Department of Genetic Medicine and Development, University Medical Centre, Geneva, Switzerland
The authors wish it to be known that, in their opinion, the first two authors should be regarded as the joint First Authors.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. A. Stewart, J. L. Campbell, and R. A. Bambara Dna2 is a structure-specific nuclease, with affinity for 5'-flap intermediates Nucleic Acids Res., November 24, 2009; (2009) gkp1055v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Stewart, J. L. Campbell, and R. A. Bambara Significance of the Dissociation of Dna2 by Flap Endonuclease 1 to Okazaki Fragment Processing in Saccharomyces cerevisiae J. Biol. Chem., March 27, 2009; 284(13): 8283 - 8291. [Abstract] [Full Text] [PDF]
|
|