Molecular basis for DNA repair synthesis on short gaps by mycobacterial Primase-Polymerase C

Nigel C Brissett,Małgorzata Korycka-Machała,Anna Brzostek,Przemysław Płociński,Katerina Zabrady,Julie Bianchi,Jarosław Dziadek,Aidan J Doherty

doi:10.1038/s41467-020-18012-8

Abstract

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol’s function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC’s gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC’s gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.

Highlights

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability
In conjunction with supporting biochemical analysis, we describe the structural elements, conformational steps and the underlying catalytic mechanism Prim–PolC employs to simultaneously engage with both sides of a gap, whilst extending the 3′ primer strand to fill in short-gapped DNA intermediates with ribonucleotides during excision repair
We reported that Prim–PolC preferentially binds to and fills in short DNA gaps with ribonucleotides during excision repair[7]

Summary

Introduction

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. To understand the molecular basis of Prim-PolC’s gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). To understand the molecular basis for its gap-binding and synthesis activities, we elucidated the crystal structures of preand post-catalytic intermediates of Prim–PolC bound to DNA substrates containing a two-nucleotide gap Based on these structures, in conjunction with supporting biochemical analysis, we describe the structural elements, conformational steps and the underlying catalytic mechanism Prim–PolC employs to simultaneously engage with both sides of a gap, whilst extending the 3′ primer strand to fill in short-gapped DNA intermediates with ribonucleotides during excision repair

Methods

Results

Conclusion