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Direct visualization of joining of DNA fragments by LigIIIβ using atomic force microscopy

Journal Article


Abstract


  • DNA ligaseIII is one of three mammalian DNA ligases (LigI, LigIII and LigIV). LigIII is distinguished from the other ligases by the presence of a Zinc finger(ZnF) that improves ligation efficiency. Previously, it was demonstrated that LigIII has two different DNA binding modules (ZnF-DBD and NTase-OB) and a jack-knife model has been proposed to explain nick recognition and joining of

    double-strand breaks. However, the oligomeric state of LigIII in solution and during ligation, and the role of ZnF in end-end ligation are unknown. Using atomic force microscopy, we directly visualized a germ cell-specific form of DNA LigIII, LigIIIb and a delZnF mutant, their interactions with DNA and ligation products. We found no evidence for oligomerization of WT and delZnF LigIIIb in solution or when complexed to DNA. Importantly, WT and delZnF proteins exist in three distinct conformational states: closed, semi-extended, and extended conformations. While WTLigIIIb protein accesses all three conformational states significantly, delZnF LigIIIb occupies primarily the closed and semi-extended states, suggesting that ZnF is part of one wing as proposed in the jack-knife model. Furthermore, binding and ligation studies on nicked and non-nicked blunt-end DNA and DNA with 50 overhang indicate that in addition to tandem joining of two linear DNA molecules, LigIIIb can mediate the ligation of a variety of higher order structures including three way junctions. In addition, with 50 overhang DNA, compared to WT protein, delZnF promotes a small but significant occurrence of three-way junctions, lassoes and knots in the presence of MgCl2. These data suggest that monomeric DNA LigIIIb is capable of binding two DNA molecules simultaneously. Currently, we are

    testing the hypothesis that the ZnF in LigIIIb may be involved in quality control ensuring only tandem end-end ligation.

UOW Authors


  •   Ghodke, Harshad
  •   Wang, Hong (external author)
  •   Cotner-Gohara, Elizabeth (external author)
  •   Kim, In-Kwon (external author)
  •   Ellenberger, Tom (external author)
  •   Van Houten, Bennett (external author)

Publication Date


  • 2010

Citation


  • Ghodke, H., Wang, H., Cotner-Gohara, E., Kim, I., Ellenberger, T. & Van Houten, B. (2010). Direct visualization of joining of DNA fragments by LigIIIβ using atomic force microscopy. Biophysical Journal, 98 (3, Supplement 1), 67a.

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/4254

Start Page


  • 67a

Volume


  • 98

Issue


  • 3, Supplement 1

Abstract


  • DNA ligaseIII is one of three mammalian DNA ligases (LigI, LigIII and LigIV). LigIII is distinguished from the other ligases by the presence of a Zinc finger(ZnF) that improves ligation efficiency. Previously, it was demonstrated that LigIII has two different DNA binding modules (ZnF-DBD and NTase-OB) and a jack-knife model has been proposed to explain nick recognition and joining of

    double-strand breaks. However, the oligomeric state of LigIII in solution and during ligation, and the role of ZnF in end-end ligation are unknown. Using atomic force microscopy, we directly visualized a germ cell-specific form of DNA LigIII, LigIIIb and a delZnF mutant, their interactions with DNA and ligation products. We found no evidence for oligomerization of WT and delZnF LigIIIb in solution or when complexed to DNA. Importantly, WT and delZnF proteins exist in three distinct conformational states: closed, semi-extended, and extended conformations. While WTLigIIIb protein accesses all three conformational states significantly, delZnF LigIIIb occupies primarily the closed and semi-extended states, suggesting that ZnF is part of one wing as proposed in the jack-knife model. Furthermore, binding and ligation studies on nicked and non-nicked blunt-end DNA and DNA with 50 overhang indicate that in addition to tandem joining of two linear DNA molecules, LigIIIb can mediate the ligation of a variety of higher order structures including three way junctions. In addition, with 50 overhang DNA, compared to WT protein, delZnF promotes a small but significant occurrence of three-way junctions, lassoes and knots in the presence of MgCl2. These data suggest that monomeric DNA LigIIIb is capable of binding two DNA molecules simultaneously. Currently, we are

    testing the hypothesis that the ZnF in LigIIIb may be involved in quality control ensuring only tandem end-end ligation.

UOW Authors


  •   Ghodke, Harshad
  •   Wang, Hong (external author)
  •   Cotner-Gohara, Elizabeth (external author)
  •   Kim, In-Kwon (external author)
  •   Ellenberger, Tom (external author)
  •   Van Houten, Bennett (external author)

Publication Date


  • 2010

Citation


  • Ghodke, H., Wang, H., Cotner-Gohara, E., Kim, I., Ellenberger, T. & Van Houten, B. (2010). Direct visualization of joining of DNA fragments by LigIIIβ using atomic force microscopy. Biophysical Journal, 98 (3, Supplement 1), 67a.

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/4254

Start Page


  • 67a

Volume


  • 98

Issue


  • 3, Supplement 1