Ultrafast repair dynamics of CPD photolesion isosteres by DNA photolyase.

Abstract

UV-B rays from sunlight can cause damage to DNA by inducing photolesions, two major forms being the cyclobutane pyrimidine dimer (CPD) and a less frequently formed 6-4 pyrimidine pyrimidone (64PP) photoproduct. Photolyases (PLs) are flavoenzymes which identify these photolesions and repair them using blue light. CPD PLs utilize 10 ultrafast elementary steps, including 7 electron transfer (ET) steps, to complete the photorepair cycle and modulate the quantum repair yield1. This can be influenced by the interplay between various molecular factors at the active site: amino acid residues, the bound substrate, solvation activity and plasticity/flexibility. Here, we have utilized a combination of steady-state kinetic studies, ultrafast laser spectroscopy and molecular dynamics simulations to approach the role of chemical properties of the substrate and binding configuration. We have measured the binding constants, quantum repair yields and ultrafast reaction rates of each elementary step involved for photorepair of 3 novel CPD isosteres (CPD1 and CPD2: 5’-Thymine<>Toluene-3’ adducts2; CPD3: T<>T dimer with 5’ pyranose sugar instead of furanose) by EcPL. We have found that EcPL can repair all 3 isosteres, despite weaker binding, albeit with lower repair yields. The enzyme utilizes universally conserved pathways to repair the isosteres. The binding configuration and redox properties of the substrate may play a direct role in modulating the reaction rates of intermolecular ET and intramolecular bond splitting steps in the repair photocycle, which in turn determines their overall repair quantum yield.