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Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme-The spore photoproduct lyase.

ABSTRACT: DNA repair enzymes typically recognize their substrate lesions with high affinity to ensure efficient lesion repair. In UV irradiated endospores, a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, termed the spore photoproduct (SP), is the dominant DNA photolesion, which is rapidly repaired during spore outgrowth mainly by spore photoproduct lyase (SPL) using an unprecedented protein-harbored radical transfer process. Surprisingly, our in vitro studies using SP-containing short oligonucleotides, pUC 18 plasmid DNA, and E. coli genomic DNA found that they are all poor substrates for SPL in general, exhibiting turnover numbers of 0.01-0.2min-1. The faster turnover numbers are reached under single turnover conditions, and SPL activity is low with oligonucleotide substrates at higher concentrations. Moreover, SP-containing oligonucleotides do not go past one turnover. In contrast, the dinucleotide SP TpT exhibits a turnover number of 0.3-0.4min-1, and the reaction may reach up to 10 turnovers. These observations distinguish SPL from other specialized DNA repair enzymes. To the best of our knowledge, SPL represents an unprecedented example of a major DNA repair enzyme that cannot effectively repair its substrate lesion within the normal DNA conformation adopted in growing cells. Factors such as other DNA binding proteins, helicases or an altered DNA conformation may cooperate with SPL to enable efficient SP repair in germinating spores. Therefore, both SP formation and SP repair are likely to be tightly controlled by the unique cellular environment in dormant and outgrowing spore-forming bacteria, and thus SP repair may be extremely slow in non-spore-forming organisms.

SUBMITTER: Yang L

PROVIDER: S-EPMC5654379 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme-The spore photoproduct lyase.

Yang Linlin L Jian Yajun Y Setlow Peter P Li Lei L

DNA repair 20170306

DNA repair enzymes typically recognize their substrate lesions with high affinity to ensure efficient lesion repair. In UV irradiated endospores, a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, termed the spore photoproduct (SP), is the dominant DNA photolesion, which is rapidly repaired during spore outgrowth mainly by spore photoproduct lyase (SPL) using an unprecedented protein-harbored radical transfer process. Surprisingly, our in vitro studies using SP-containing short oligonucleot ...[more]

PMID: 28320593

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Project description:5-Thyminyl-5,6-dihydrothymine (also called spore photoproduct or SP) is the exclusive DNA photodamage product in bacterial endospores. It is repaired by a radical SAM (S-adenosylmethionine) enzyme, the spore photoproduct lyase (SPL), at the bacterial early germination phase. Our previous studies proved that SPL utilizes the 5'-dA• generated by the SAM cleavage reaction to abstract the H(6proR) atom to initiate the SP repair process. The resulting thymine allylic radical was suggested to take an H atom from an unknown protein source, most likely cysteine 141. Here we show that C141 can be readily alkylated in the native SPL by an iodoacetamide treatment, suggesting that it is accessible to the TpT radical. SP repair by the SPL C141A mutant yields TpTSO(2)(-) and TpT simultaneously from the very beginning of the reaction; no lag phase is observed for TpTSO(2)(-) formation. Should any other protein residue serve as the H donor, its presence would result in TpT being the major product at least for the first enzyme turnover. These observations provide strong evidence to support C141 as the direct H atom donor. Moreover, because of the lack of this intrinsic H donor, the C141A mutant produces TpT via an unprecedented thymine cation radical reduction (proton-coupled electron transfer) process, contrasting to the H atom transfer mechanism in the wild-type (WT) SPL reaction. The C141A mutant repairs SP at a rate that is ~3-fold slower than that of the WT enzyme. Formation of TpTSO(2)(-) and TpT exhibits a V(max) deuterium kinetic isotope effect (KIE) of 1.7 ± 0.2, which is smaller than the (D)V(max) KIE of 2.8 ± 0.3 determined for the WT SPL reaction. These findings suggest that removing the intrinsic H atom donor disturbs the rate-limiting process during enzyme catalysis. As expected, the prereduced C141A mutant supports only ~0.4 turnover, which is in sharp contrast to the >5 turnovers exhibited by the WT SPL reaction, suggesting that the enzyme catalytic cycle (SAM regeneration) is disrupted by this single mutation.

Dataset Information

Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme-The spore photoproduct lyase.

Publications

Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme-The spore photoproduct lyase.

Similar Datasets

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