In:
Science, American Association for the Advancement of Science (AAAS), Vol. 382, No. 6674 ( 2023-12)
Abstract:
Dimerization of thymine bases to form a cyclobutane-pyrimidine dimer (CPD) is a common ultraviolet light–induced DNA lesion. Photolyases catalyze light-triggered repair of CPD-DNA, thus contributing to genome stability in many organisms. Combining time-resolved crystallography and computational analyses, we report an atomic visualization of the photolyase-catalyzed DNA repair process. We captured electron transfer at low picoseconds, chemical steps at picoseconds to nanoseconds, active-site recovery at nanoseconds to microseconds, and reannealing of the double-stranded DNA (dsDNA) at submilliseconds, forging new ground in DNA repair, structural biology, and enzymology. RATIONALE Mechanistic models from previous spectroscopic studies set a framework for using time-resolved serial femtosecond crystallography (TR-SFX) to visualize this catalytic process. This technique provides a concise view of not only the repair chemistry but also hitherto unknown postrepair events. RESULTS Two series of TR-SFX experiments were performed, one from picoseconds to nanoseconds and the other from nanoseconds to microseconds (see the figure). Our visualization of the repair of a CPD begins at 100 ps, with Arg 256 (R256) becoming dynamic and moving to stabilize the CPD, which suggests the initiation of the forward electron transfer from the reduced, anionic hydroquinone state of flavin adenine dinucleotide (FADH − ) to the CPD. At 650 ps, the C5–C5′ bond of the CPD is predominantly split, and at 1 ns, the C6–C6′ is likewise split. Recovery of R256, a five-water cluster (5WC), and the FADH − coenzyme occurs during the next 500 ns, returning to their respective resting-state conformations. The repaired thymine bases remain in the active site during this time and then start to return to reanneal with the dsDNA in the microsecond range. The 200-μs structures show the coexistence of a back-flipping intermediate and the reannealed product before its final release from the enzyme. CONCLUSION Our results uncover the atomic mechanism of how DNA photolyases repair DNA in real time. These data reveal an ordered breaking of the covalent C–C bonds and opening of the cyclobutane ring, as well as the concomitant conformational changes of the photolyase and its FAD coenzyme. Defined intermediates were also captured when the enzyme-product complex was recovering and repaired product bases were departing from the active site to pair with their complementary bases in the dsDNA. Elucidation of the main processes and key intermediates in the DNA repair reaction catalyzed by photolyase. Selected intermediates (cyan) of the repair process are overlaid with the structure of the dark state (gray) to illustrate structural changes during catalysis. T7 and T8 are the thymine-7 and thymine-8, the damaged 5′- and 3′-thymines of the CPD lesion in the DNA strand. TT refers to the two thymines together.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.add7795
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2023
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
SSG:
11
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