Abstract
The multi-step base excision repair (BER) pathway is initiated by a set of enzymes, known as DNA glycosylases, able to scan DNA and detect modified bases among a vast number of normal bases. While DNA glycosylases in the BER pathway generally bend the DNA and flip damaged bases into lesion specific pockets, the HEAT-like repeat DNA glycosylase AlkD detects and excises bases without sequestering the base from the DNA helix. We show by single-molecule tracking experiments that AlkD scans DNA without forming a stable interrogation complex. This contrasts with previously studied repair enzymes that need to flip bases into lesion-recognition pockets and form stable interrogation complexes. Moreover, we show by design of a loss-of-function mutant that the bimodality in scanning observed for the structural homologue AlkF is due to a key structural differentiator between AlkD and AlkF; a positively charged β-hairpin able to protrude into the major groove of DNA.
Highlights
The multi-step base excision repair (BER) pathway is initiated by a set of enzymes, known as DNA glycosylases, able to scan DNA and detect modified bases among a vast number of normal bases
DNA glycosylases can be classified into the structural superfamilies helix–hairpin–helix (HhH), helix-two-turn-helix (H2TH), and HEAT-like repeat (HLR), as well as the uracil-DNA glycosylase (UNG) and alkyladenine-DNA glycosylase (AAG) families[1,2]
Four of these are present in human cells, while the HEAT-like repeat family has only been detected in bacteria, archaea, and some simple uni- and multicellular eukaryotes[3,4,5,6]
Summary
The multi-step base excision repair (BER) pathway is initiated by a set of enzymes, known as DNA glycosylases, able to scan DNA and detect modified bases among a vast number of normal bases. Classification of the trajectories into two modes using HMM analysis (Fig. 2d, orange and blue histograms) indicates the existence of an intermediate state in DNA scanning within an apparent single-mode distribution of the overall diffusion rate of AlkD (Fig. 2d, red curve).
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