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Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

Journal Article


Abstract


  • How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3–0.8, 8.1, and 113–126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism.

UOW Authors


  •   Ghodke, Harshad (external author)
  •   Wang, Hong (external author)
  •   Hsieh, Ching L. (external author)
  •   Woldemeskel, Selamawit (external author)
  •   Watkins, Simon (external author)
  •   Rapic-Otrin, Vesna (external author)
  •   Van Houten, Bennett (external author)

Publication Date


  • 2014

Citation


  • Ghodke, H., Wang, H., Hsieh, C. L., Woldemeskel, S., Watkins, S. C., Rapic-Otrin, V. & Van Houten, B. (2014). Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates. Proceedings of the National Academy of Sciences of USA, 111 (18), E1862-E1871.

Scopus Eid


  • 2-s2.0-84899825410

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/4247

Start Page


  • E1862

End Page


  • E1871

Volume


  • 111

Issue


  • 18

Abstract


  • How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3–0.8, 8.1, and 113–126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism.

UOW Authors


  •   Ghodke, Harshad (external author)
  •   Wang, Hong (external author)
  •   Hsieh, Ching L. (external author)
  •   Woldemeskel, Selamawit (external author)
  •   Watkins, Simon (external author)
  •   Rapic-Otrin, Vesna (external author)
  •   Van Houten, Bennett (external author)

Publication Date


  • 2014

Citation


  • Ghodke, H., Wang, H., Hsieh, C. L., Woldemeskel, S., Watkins, S. C., Rapic-Otrin, V. & Van Houten, B. (2014). Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates. Proceedings of the National Academy of Sciences of USA, 111 (18), E1862-E1871.

Scopus Eid


  • 2-s2.0-84899825410

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/4247

Start Page


  • E1862

End Page


  • E1871

Volume


  • 111

Issue


  • 18