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PMID: 11438646 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Identification of rad27 mutations that confer differential defects in mutation avoidance, repeat tract instability, and flap cleavage.

Molecular and cellular biology ·Vol. 21 ·No. 15 ·2001-08-00 ·Pages 4889-99

Xie Y, Liu Y, Argueso JL, Henricksen LA, Kao HI, Bambara RA, Alani E

Abstract

In eukaryotes, the nuclease activity of Rad27p (Fen1p) is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5' ends of Okazaki fragments. Genetic analysis of Saccharomyces cerevisiae also has identified a role for Rad27p in mutation avoidance. rad27Delta mutants display both a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result primarily from duplications of DNA sequences that are flanked by direct repeats. These observations suggested that Rad27p activities in DNA replication and repair could be altered by mutagenesis and specifically assayed. To test this idea, we analyzed two rad27 alleles, rad27-G67S and rad27-G240D, that were identified in a screen for mutants that displayed repeat tract instability and mutator phenotypes. In chromosome stability assays, rad27-G67S strains displayed a higher frequency of repeat tract instabilities relative to CAN1 duplication events; in contrast, the rad27-G240D strains displayed the opposite phenotype. In biochemical assays, rad27-G67Sp displayed a weak exonuclease activity but significant single- and double-flap endonuclease activities. In contrast, rad27-G240Dp displayed a significant double-flap endonuclease activity but was devoid of exonuclease activity and showed only a weak single-flap endonuclease activity. Based on these observations, we hypothesize that the rad27-G67S mutant phenotypes resulted largely from specific defects in nuclease function that are important for degrading bubble intermediates, which can lead to DNA slippage events. The rad27-G240D mutant phenotypes were more difficult to reconcile to a specific biochemical defect, suggesting a structural role for Rad27p in DNA replication and repair. Since the mutants provide the means to relate nuclease functions in vitro to genetic characteristics in vivo, they are valuable tools for further analyses of the diverse biological roles of Rad27p.

MeSH Terms
Alleles Canavanine/pharmacology DNA Repair Dose-Response Relationship, Drug Drug Resistance/genetics Endodeoxyribonucleases/chemistry,genetics,metabolism Flap Endonucleases Genotype Models, Genetic Mutation Phenotype Protein Binding Repetitive Sequences, Nucleic Acid Saccharomyces cerevisiae/genetics,metabolism Saccharomyces cerevisiae Proteins
Chemicals
Saccharomyces cerevisiae Proteins Canavanine Endodeoxyribonucleases Flap Endonucleases RAD27 protein, S cerevisiae
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Xie Y
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
Liu Y
Argueso J L
Henricksen L A
Kao H I
Bambara R A
Alani E
References (55)
55 references, click to expand
  1. Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA.
    Science. 1995 Jul 14;269(5221):238-40 PMID: 7618086
  2. Mutations in RAD27 define a potential link between G1 cyclins and DNA replication.
    Mol Cell Biol. 1995 Aug;15(8):4291-302 PMID: 7623823
  3. Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen.
    J Biol Chem. 1995 Sep 22;270(38):22109-12 PMID: 7673186
  4. Calf 5' to 3' exo/endonuclease must slide from a 5' end of the substrate to perform structure-specific cleavage.
    J Biol Chem. 1995 Dec 22;270(51):30377-83 PMID: 8530463
  5. Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair.
    Genes Dev. 1996 Feb 15;10(4):407-20 PMID: 8600025
  6. Requirement of the yeast MSH3 and MSH6 genes for MSH2-dependent genomic stability.
    J Biol Chem. 1996 Mar 29;271(13):7285-8 PMID: 8631743
  7. Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA.
    Nucleic Acids Res. 1996 Jun 1;24(11):2036-43 PMID: 8668533
  8. Structure of bacteriophage T4 RNase H, a 5' to 3' RNA-DNA and DNA-DNA exonuclease with sequence similarity to the RAD2 family of eukaryotic proteins.
    Cell. 1996 Jun 28;85(7):1101-12 PMID: 8674116
  9. A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease.
    Nature. 1996 Jul 4;382(6586):90-3 PMID: 8657312
  10. Role of calf RTH-1 nuclease in removal of 5'-ribonucleotides during Okazaki fragment processing.
    Biochemistry. 1996 Jul 16;35(28):9266-77 PMID: 8703932
  11. p21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication complex.
    Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11597-602 PMID: 8876181
  12. A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair.
    Cell. 1997 Jan 24;88(2):253-63 PMID: 9008166
  13. A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function.
    Mol Cell Biol. 1997 Apr;17(4):2136-42 PMID: 9121462
  14. The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair.
    Bioessays. 1997 Mar;19(3):233-40 PMID: 9080773
  15. Enzymes and reactions at the eukaryotic DNA replication fork.
    J Biol Chem. 1997 Feb 21;272(8):4647-50 PMID: 9081985
  16. Repeat expansion--all in a flap?
    Nat Genet. 1997 Jun;16(2):116-8 PMID: 9171819
  17. Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2.
    Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7487-92 PMID: 9207118
  18. Functional analysis of point mutations in human flap endonuclease-1 active site.
    Nucleic Acids Res. 1997 Aug 15;25(16):3332-8 PMID: 9241249
  19. Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen.
    EMBO J. 1998 Apr 15;17(8):2412-25 PMID: 9545252
  20. Destabilization of yeast micro- and minisatellite DNA sequences by mutations affecting a nuclease involved in Okazaki fragment processing (rad27) and DNA polymerase delta (pol3-t).
    Mol Cell Biol. 1998 May;18(5):2779-88 PMID: 9566897
  21. The crystal structure of flap endonuclease-1 from Methanococcus jannaschii.
    Nat Struct Biol. 1998 Aug;5(8):707-13 PMID: 9699635
  22. Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity.
    Cell. 1998 Oct 2;95(1):135-46 PMID: 9778254
  23. Homologous recombination is required for the viability of rad27 mutants.
    Nucleic Acids Res. 1998 Dec 15;26(24):5589-95 PMID: 9837987
  24. Characterization of the repeat-tract instability and mutator phenotypes conferred by a Tn3 insertion in RFC1, the large subunit of the yeast clamp loader.
    Genetics. 1999 Feb;151(2):499-509 PMID: 9927446
  25. Human exonuclease 1 functionally complements its yeast homologues in DNA recombination, RNA primer removal, and mutation avoidance.
    J Biol Chem. 1999 Jun 18;274(25):17893-900 PMID: 10364235
  26. Thermostable flap endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically.
    J Biol Chem. 1999 Jun 25;274(26):18297-309 PMID: 10373433
  27. A novel role in DNA metabolism for the binding of Fen1/Rad27 to PCNA and implications for genetic risk.
    Mol Cell Biol. 1999 Aug;19(8):5373-82 PMID: 10409728
  28. Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1.
    J Biol Chem. 2000 Apr 7;275(14):10498-505 PMID: 10744741
  29. BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures.
    Genes Dev. 2000 Apr 15;14(8):927-39 PMID: 10783165
  30. Inhibition of flap endonuclease 1 by flap secondary structure and relevance to repeat sequence expansion.
    J Biol Chem. 2000 Jun 2;275(22):16420-7 PMID: 10748145
  31. Two modes of FEN1 binding to PCNA regulated by DNA.
    EMBO J. 2000 Jul 17;19(14):3811-21 PMID: 10899134
  32. Cell cycle-dependent and DNA damage-inducible nuclear localization of FEN-1 nuclease is consistent with its dual functions in DNA replication and repair.
    J Biol Chem. 2001 Feb 16;276(7):4901-8 PMID: 11053418
  33. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance.
    Mol Gen Genet. 1984;197(2):345-6 PMID: 6394957
  34. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.
    Science. 1985 Dec 20;230(4732):1350-4 PMID: 2999980
  35. A rapid, efficient method for isolating DNA from yeast.
    Gene. 1986;42(2):169-73 PMID: 3015730
  36. Initiation of adenovirus DNA replication. I. Mechanism of action of a host protein required for replication of adenovirus DNA templates devoid of the terminal protein.
    J Biol Chem. 1988 Jul 15;263(20):9801-8 PMID: 2838479
  37. Complete enzymatic synthesis of DNA containing the SV40 origin of replication.
    J Biol Chem. 1988 Dec 25;263(36):19723-33 PMID: 2848839
  38. Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
    Gene. 1989 Apr 15;77(1):51-9 PMID: 2744487
  39. Discontinuous DNA synthesis by purified mammalian proteins.
    J Biol Chem. 1990 Oct 25;265(30):18461-71 PMID: 2170412
  40. Repair of specific base pair mismatches formed during meiotic recombination in the yeast Saccharomyces cerevisiae.
    Mol Cell Biol. 1991 Feb;11(2):737-45 PMID: 1990280
  41. Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier.
    Yeast. 1991 Apr;7(3):253-63 PMID: 1882550
  42. Instability of simple sequence DNA in Saccharomyces cerevisiae.
    Mol Cell Biol. 1992 Jun;12(6):2749-57 PMID: 1588966
  43. Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mitochondrial and nuclear functions.
    Genetics. 1992 Dec;132(4):975-85 PMID: 1334021
  44. Completion of mammalian lagging strand DNA replication using purified proteins.
    J Biol Chem. 1993 Jul 15;268(20):15136-41 PMID: 8392066
  45. Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair.
    Nature. 1993 Sep 16;365(6443):274-6 PMID: 8371783
  46. The characterization of a mammalian DNA structure-specific endonuclease.
    EMBO J. 1994 Mar 1;13(5):1235-46 PMID: 8131753
  47. Reconstitution of complete SV40 DNA replication with purified replication factors.
    J Biol Chem. 1994 Apr 8;269(14):10923-34 PMID: 8144677
  48. Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair.
    Genes Dev. 1994 Jun 1;8(11):1344-55 PMID: 7926735
  49. Enzymatic completion of mammalian lagging-strand DNA replication.
    Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9803-7 PMID: 7524089
  50. Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene.
    J Bacteriol. 1995 Jan;177(2):364-71 PMID: 7814325
  51. A role for exonuclease I from S. pombe in mutation avoidance and mismatch correction.
    Science. 1995 Feb 24;267(5201):1166-9 PMID: 7855597
  52. Conditional lethality of null mutations in RTH1 that encodes the yeast counterpart of a mammalian 5'- to 3'-exonuclease required for lagging strand DNA synthesis in reconstituted systems.
    J Biol Chem. 1995 Mar 3;270(9):4193-6 PMID: 7876174
  53. DNA structural elements required for FEN-1 binding.
    J Biol Chem. 1995 Mar 3;270(9):4503-8 PMID: 7876218
  54. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C.
    Yeast. 1995 Jan;11(1):53-5 PMID: 7762301
  55. Separation-of-function mutations in Saccharomyces cerevisiae MSH2 that confer mismatch repair defects but do not affect nonhomologous-tail removal during recombination.
    Mol Cell Biol. 1999 Nov;19(11):7558-67 PMID: 10523644
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.21.15.4889-4899.2001
Published
2001-08-00
Pages
4889-99
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC87203
Subset
IM
Grants
NIGMS NIH HHS · R01 GM024441 · United States
NIGMS NIH HHS · F32 GM018961 · United States
NIGMS NIH HHS · GM18961 · United States
NIGMS NIH HHS · GM24441 · United States
NIGMS NIH HHS · R01 GM053085 · United States
NIGMS NIH HHS · GM53085 · United States
External Links
PubMed source DOI: 10.1128/MCB.21.15.4889-4899.2001 PMC Full Text View journal details
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