Displacement of Slow-Turnover DNA Glycosylases by Molecular Traffic on DNA.

Anna V Yudkina,Anna V Yudkina,Anna V Yudkina,Nina A Moor,Nina A Moor,Dmitry O Zharkov,Dmitry O Zharkov,Dmitry O Zharkov,Eugenia A Diatlova,Anton V Endutkin,Anton V Endutkin,Inga R Grin,Inga R Grin,Inga R Grin,Ivan P Vokhtantsev,Ivan P Vokhtantsev,Ivan P Vokhtantsev

doi:10.3390/genes11080866

Abstract

In the base excision repair pathway, the initiating enzymes, DNA glycosylases, remove damaged bases and form long-living complexes with the abasic DNA product, but can be displaced by AP endonucleases. However, many nuclear proteins can move along DNA, either actively (such as DNA or RNA polymerases) or by passive one-dimensional diffusion. In most cases, it is not clear whether this movement is disturbed by other bound proteins or how collisions with moving proteins affect the bound proteins, including DNA glycosylases. We have used a two-substrate system to study the displacement of human OGG1 and NEIL1 DNA glycosylases by DNA polymerases in both elongation and diffusion mode and by D4, a passively diffusing subunit of a viral DNA polymerase. The OGG1–DNA product complex was disrupted by DNA polymerase β (POLβ) in both elongation and diffusion mode, Klenow fragment (KF) in the elongation mode and by D4. NEIL1, which has a shorter half-life on DNA, was displaced more efficiently. Hence, both possibly specific interactions with POLβ and nonspecific collisions (KF, D4) can displace DNA glycosylases from DNA. The protein movement along DNA was blocked by very tightly bound Cas9 RNA-targeted nuclease, providing an upper limit on the efficiency of obstacle clearance.

Highlights

Genomic DNA is bound to a number of structural, regulatory and catalytic proteins, some of which form very strong non-covalent complexes with DNA
We address the displacement of a slow-turnover 8-oxoguanine–DNA glycosylase (OGG1) by DNA polymerase β, which may be specific since both proteins belong to the human Base excision DNA repair (BER) system and by the Klenow fragment of Escherichia coli DNA polymerase I (KF) and vaccinia virus D4 protein, used here as models of nonspecific DNA-directed interactions
OGG1 Can Be Displaced by DNA Polymerase β

Summary

Introduction

Genomic DNA is bound to a number of structural, regulatory and catalytic proteins, some of which form very strong non-covalent complexes with DNA. There is a variety of nonstructural proteins tightly bound to DNA. Transcription and replication elongation complexes unwind the helix and proceed actively displacing some protein obstacles but may be blocked by others [4]. Many proteins have been shown to move along DNA non-directionally by facilitated diffusion [5,6], and it is generally not known how they move over roadblocks presented by tightly bound molecules of other proteins. The competition between proteins stably bound to DNA and moving along it is sometimes referred to as “molecular traffic jam” on DNA [4]. Given the diversity of pathways in which DNA-bound proteins participate, most of collisions on DNA are expected to

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Conclusion