title: Functional mapping of the fission yeast DNA polymerase delta B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesis.
abstract: BACKGROUND
  DNA polymerase delta plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol delta is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol delta comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively.
  
  
  RESULTS
  To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1.
  
  
  CONCLUSIONS
  In the absence of a three-dimensional structure of the entire Pol delta complex, the results of this study highlight regions in Cdc1 that are vital for protein function in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK. sanchezgarciajavier@yahoo.com
authors.forename: Javier
authors.initials: J
authors.lastname: Sanchez Garcia
authors.forename: Andrey G
authors.initials: AG
authors.lastname: Baranovskiy
authors.forename: Elena V
authors.initials: EV
authors.lastname: Knatko
authors.forename: Fiona C
authors.initials: FC
authors.lastname: Gray
authors.forename: Tahir H
authors.initials: TH
authors.lastname: Tahirov
authors.forename: Stuart A
authors.initials: SA
authors.lastname: MacNeill
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D021122:Protein Subunits; D004258:DNA Polymerase III
references.citation: Crit Rev Biochem Mol Biol. 2005 Mar-Apr;40(2):115-28
references.pmid: 15814431
references.citation: Trends Biochem Sci. 2001 Jan;26(1):16-7
references.pmid: 11165510
references.citation: Mol Cell. 2008 May 9;30(3):259-60
references.pmid: 18471969
references.citation: Nucleic Acids Res. 1998 Jan 15;26(2):477-85
references.pmid: 9421503
references.citation: EMBO J. 2009 Jul 8;28(13):1978-87
references.pmid: 19494830
references.citation: EMBO J. 1996 Sep 2;15(17):4613-28
references.pmid: 8887553
references.citation: Science. 2007 Jul 6;317(5834):127-30
references.pmid: 17615360
references.citation: Nucleic Acids Res. 2008 Sep;36(15):5102-10
references.pmid: 18676977
references.citation: Biochemistry. 2002 Nov 5;41(44):13133-42
references.pmid: 12403614
references.citation: Trends Microbiol. 2000 Dec;8(12):571-7
references.pmid: 11115754
references.citation: Nucleic Acids Res. 1992 Feb 11;20(3):621
references.pmid: 1741305
references.citation: J Biol Chem. 2006 May 26;281(21):14748-55
references.pmid: 16510448
references.citation: Gene. 1999 Apr 1;230(1):15-22
references.pmid: 10196469
references.citation: EMBO J. 1999 Nov 15;18(22):6339-48
references.pmid: 10562546
references.citation: Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11244-9
references.pmid: 9326594
references.citation: Methods Enzymol. 1991;194:795-823
references.pmid: 2005825
references.citation: J Mol Biol. 1997 Nov 21;274(1):39-53
references.pmid: 9398514
references.citation: Mol Cell. 2008 Apr 25;30(2):137-44
references.pmid: 18439893
references.citation: Biochim Biophys Acta. 2003 Sep 23;1651(1-2):163-71
references.pmid: 14499601
references.citation: Nucleic Acids Res. 1998 Aug 15;26(16):3746-52
references.pmid: 9685491
references.citation: J Biol Chem. 2002 Feb 8;277(6):3894-901
references.pmid: 11711545
references.citation: Mol Biol Cell. 1994 Oct;5(10):1145-58
references.pmid: 7865880
references.citation: Nucleic Acids Res. 2004 Dec 02;32(21):6367-77
references.pmid: 15576681
references.citation: Nucleic Acids Res. 1993 Aug 11;21(16):3821-8
references.pmid: 8367300
references.citation: Curr Genet. 2000 Mar;37(3):159-67
references.pmid: 10794172
references.citation: J Biol Chem. 2002 Sep 27;277(39):36853-62
references.pmid: 12124382
references.citation: Mol Cell. 1999 May;3(5):679-85
references.pmid: 10360184
references.citation: BMC Mol Biol. 2004 Dec 03;5(1):21
references.pmid: 15579205
references.citation: Nucleic Acids Res. 2004 Jun 01;32(10):3005-16
references.pmid: 15173383
references.citation: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Sep 1;64(Pt 9):822-4
references.pmid: 18765914
references.citation: Nat Struct Mol Biol. 2006 Jan;13(1):35-43
references.pmid: 16369485
references.citation: Mol Gen Genet. 1976 Jul 23;146(2):167-78
references.pmid: 958201
references.citation: Mol Gen Genet. 1997 Mar 18;254(1):93-7
references.pmid: 9108295
references.citation: Curr Genet. 1998 Oct;34(4):250-8
references.pmid: 9799358
references.citation: Mol Gen Genet. 1981;182(1):119-24
references.pmid: 6943408
references.citation: Mol Gen Genet. 1995 Jan 20;246(2):212-22
references.pmid: 7862092
references.citation: Nat Rev Microbiol. 2008 Mar;6(3):245-52
references.pmid: 18274537
references.citation: J Biol Chem. 1997 Nov 14;272(46):28833-6
references.pmid: 9360947
references.citation: FEBS J. 2009 Sep;276(17):4803-13
references.pmid: 19664060
references.citation: Nucleic Acids Res. 1997 May 1;25(9):1866-7
references.pmid: 9108174
references.citation: Trends Biochem Sci. 1999 Jan;24(1):14-6
references.pmid: 10087916
references.citation: Nucleic Acids Res. 2005 Jul 21;33(13):4078-89
references.pmid: 16040599
references.citation: Nucleic Acids Res. 1995 Sep 11;23(17):3493-500
references.pmid: 7567461
references.citation: Cell Cycle. 2008 Oct;7(19):3026-36
references.pmid: 18818516
references.citation: EMBO J. 2000 Mar 1;19(5):1108-18
references.pmid: 10698951
references.citation: J Biol Chem. 2000 Feb 18;275(7):5153-62
references.pmid: 10671561
references.citation: Genetics. 1997 Aug;146(4):1239-51
references.pmid: 9258670
references.citation: Trends Cell Biol. 2008 Nov;18(11):521-7
references.pmid: 18824354