publication . Article . 2018

Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5′ exonuclease activities

Maria Loressa Uson; Ayala Carl; Yehuda Goldgur; Stewart Shuman;
Open Access English
  • Published: 09 Apr 2018 Journal: Nucleic Acids Research, volume 46, issue 8, pages 4,164-4,175 (issn: 0305-1048, eissn: 1362-4962, Copyright policy)
  • Publisher: Oxford University Press
Abstract
Abstract Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5′ exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA’s activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they...
Subjects
free text keywords: Nucleic Acid Enzymes, Genetics
Funded by
NIH| Pixel Array Detector for X-ray Crystallography
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 1S10RR029205-01
  • Funding stream: NATIONAL CENTER FOR RESEARCH RESOURCES
,
NIH| NE-CAT Center for Advanced Macromolecular Crystallography
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 4P41GM103403-14
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| MOUSE GENETICS
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 2P30CA008748-43
  • Funding stream: NATIONAL CANCER INSTITUTE
,
NIH| DNA Ligases in Mycobacterial DNA repair &Pathogenesis
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5R01AI064693-03
  • Funding stream: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
40 references, page 1 of 3

1.Finger L.D., Atack J.M., Tsutakawa S., Classen S., Tainer J., Grasby J., Shen B.The wonders of flap endonucleases: structure, function, mechanism and regulation. Subcell. Biochem.2012; 62:301–326.22918592 [OpenAIRE] [PubMed]

2.Tomlinson C.G., Atack J.M., Chapados B., Tainer J.A., Grasby J.A.Substrate recognition and catalysis by flap endonucleases and related enzymes. Biochem. Soc. Trans.2010; 38:433–437.20298197 [OpenAIRE] [PubMed]

3.Lyamichev V., Brow M.A., Dahlberg J.E.Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science. 1993; 260:778–783.7683443 [OpenAIRE] [PubMed]

4.Xu Y., Derbyshire V., Ng K., Sun X.C., Grindley N.D., Joyce C.M.Biochemical and mutational studies of the 5′-3′ exonuclease of DNA polymerase I of Escherichia coli. J. Mol. Biol.1997; 268:284–302.9159471 [PubMed]

5.Lyamichev V., Brow M.A., Varvel V.E., Dahlberg J.E.Comparison of the 5′ nuclease activities of Taq DNA polymerase and its isolated nuclease domain. Proc. Natl. Acad. Sci. U.S.A.1999; 96:6143–6148.10339555 [OpenAIRE] [PubMed]

6.Mizrahi V., Huberts P.Deoxy- and dideoxynucleotide discrimination and identification of critical 5′ nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis. Nucleic Acids Res.1996; 24:4845–4852.9016652 [OpenAIRE] [PubMed]

7.Uson M.L., Ghosh S., Shuman S.The DNA repair repertoire of Mycobacterium smegmatis FenA includes the incision of DNA 5′ flaps and the removal of 5′ adenylylated products of aborted nick ligation. J. Bacteriol.2017; 199:e00304–e00317.28630124 [OpenAIRE] [PubMed]

8.Anstey-Gilbert C.S., Hemsworth G.R., Flemming C.S., Hodskinson M.R., Zhang J., Sedelnikova S.E., Stillman T.J., Sayers J.R., Artymiuk P.J.The structure of Escherichia coli ExoIX – implications for DNA binding and catalysis in flap endonucleases. Nucleic Acids Res.2013; 41:8357–8367.23821668 [OpenAIRE] [PubMed]

9.Fukushima S., Itaya M., Kato H., Ogasawara N., Yoshikawa H.Reassessment of the in vivo function of DNA polymerase I and RNase H in bacterial cell growth. J. Bacteriol.2007; 189:8575–8583.17905985 [OpenAIRE] [PubMed]

10.Ceska T.A., Sayers J.R., Stier G., Suck D.A helical arch allowing single-stranded DNA to thread through T5 5′-exonuclease. Nature. 1996; 382:90–93.8657312 [OpenAIRE] [PubMed]

11.AlMalki F.A., Flemming C.S., Zhang J., Feng M., Sedelnikova S.E., Ceska T., Rafferty J.B., Sayers J.R., Artymiuk P.J.Direct observation of DNA threading in flap endonuclease complexes. Nat. Struct. Mol. Biol.2016; 23:640–646.27273516 [OpenAIRE] [PubMed]

12.Mueser T.C., Nossal N.G., Hyde C.C.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; 85:1101–1112.8674116 [OpenAIRE] [PubMed]

13.Devos J.M., Tomanicek S.J., Jones C.E., Nossal N.G., Mueser T.C.Crystal structure of bacteriophage T4 5′ nuclease in complex with a branched DNA reveals how flap endonuclease-1 family nucleases bind their substrates. J. Biol. Chem.2007; 282:31713–31724.17693399 [PubMed]

14.Harrington J.J., Lieber M.R.The characterization of a mammalian DNA structure-specific endonuclease. EMBO J.1994; 13:1235–1246.8131753 [OpenAIRE] [PubMed]

15.Balakrishnan L., Barbara R.A.Flap endonuclease 1. Annu. Rev. Biochem.2013; 82:119–138.23451868 [OpenAIRE] [PubMed]

40 references, page 1 of 3
Abstract
Abstract Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5′ exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA’s activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they...
Subjects
free text keywords: Nucleic Acid Enzymes, Genetics
Funded by
NIH| Pixel Array Detector for X-ray Crystallography
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 1S10RR029205-01
  • Funding stream: NATIONAL CENTER FOR RESEARCH RESOURCES
,
NIH| NE-CAT Center for Advanced Macromolecular Crystallography
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 4P41GM103403-14
  • Funding stream: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
,
NIH| MOUSE GENETICS
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 2P30CA008748-43
  • Funding stream: NATIONAL CANCER INSTITUTE
,
NIH| DNA Ligases in Mycobacterial DNA repair &Pathogenesis
Project
  • Funder: National Institutes of Health (NIH)
  • Project Code: 5R01AI064693-03
  • Funding stream: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
40 references, page 1 of 3

1.Finger L.D., Atack J.M., Tsutakawa S., Classen S., Tainer J., Grasby J., Shen B.The wonders of flap endonucleases: structure, function, mechanism and regulation. Subcell. Biochem.2012; 62:301–326.22918592 [OpenAIRE] [PubMed]

2.Tomlinson C.G., Atack J.M., Chapados B., Tainer J.A., Grasby J.A.Substrate recognition and catalysis by flap endonucleases and related enzymes. Biochem. Soc. Trans.2010; 38:433–437.20298197 [OpenAIRE] [PubMed]

3.Lyamichev V., Brow M.A., Dahlberg J.E.Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science. 1993; 260:778–783.7683443 [OpenAIRE] [PubMed]

4.Xu Y., Derbyshire V., Ng K., Sun X.C., Grindley N.D., Joyce C.M.Biochemical and mutational studies of the 5′-3′ exonuclease of DNA polymerase I of Escherichia coli. J. Mol. Biol.1997; 268:284–302.9159471 [PubMed]

5.Lyamichev V., Brow M.A., Varvel V.E., Dahlberg J.E.Comparison of the 5′ nuclease activities of Taq DNA polymerase and its isolated nuclease domain. Proc. Natl. Acad. Sci. U.S.A.1999; 96:6143–6148.10339555 [OpenAIRE] [PubMed]

6.Mizrahi V., Huberts P.Deoxy- and dideoxynucleotide discrimination and identification of critical 5′ nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis. Nucleic Acids Res.1996; 24:4845–4852.9016652 [OpenAIRE] [PubMed]

7.Uson M.L., Ghosh S., Shuman S.The DNA repair repertoire of Mycobacterium smegmatis FenA includes the incision of DNA 5′ flaps and the removal of 5′ adenylylated products of aborted nick ligation. J. Bacteriol.2017; 199:e00304–e00317.28630124 [OpenAIRE] [PubMed]

8.Anstey-Gilbert C.S., Hemsworth G.R., Flemming C.S., Hodskinson M.R., Zhang J., Sedelnikova S.E., Stillman T.J., Sayers J.R., Artymiuk P.J.The structure of Escherichia coli ExoIX – implications for DNA binding and catalysis in flap endonucleases. Nucleic Acids Res.2013; 41:8357–8367.23821668 [OpenAIRE] [PubMed]

9.Fukushima S., Itaya M., Kato H., Ogasawara N., Yoshikawa H.Reassessment of the in vivo function of DNA polymerase I and RNase H in bacterial cell growth. J. Bacteriol.2007; 189:8575–8583.17905985 [OpenAIRE] [PubMed]

10.Ceska T.A., Sayers J.R., Stier G., Suck D.A helical arch allowing single-stranded DNA to thread through T5 5′-exonuclease. Nature. 1996; 382:90–93.8657312 [OpenAIRE] [PubMed]

11.AlMalki F.A., Flemming C.S., Zhang J., Feng M., Sedelnikova S.E., Ceska T., Rafferty J.B., Sayers J.R., Artymiuk P.J.Direct observation of DNA threading in flap endonuclease complexes. Nat. Struct. Mol. Biol.2016; 23:640–646.27273516 [OpenAIRE] [PubMed]

12.Mueser T.C., Nossal N.G., Hyde C.C.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; 85:1101–1112.8674116 [OpenAIRE] [PubMed]

13.Devos J.M., Tomanicek S.J., Jones C.E., Nossal N.G., Mueser T.C.Crystal structure of bacteriophage T4 5′ nuclease in complex with a branched DNA reveals how flap endonuclease-1 family nucleases bind their substrates. J. Biol. Chem.2007; 282:31713–31724.17693399 [PubMed]

14.Harrington J.J., Lieber M.R.The characterization of a mammalian DNA structure-specific endonuclease. EMBO J.1994; 13:1235–1246.8131753 [OpenAIRE] [PubMed]

15.Balakrishnan L., Barbara R.A.Flap endonuclease 1. Annu. Rev. Biochem.2013; 82:119–138.23451868 [OpenAIRE] [PubMed]

40 references, page 1 of 3
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