Dataset Information

Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein.

ABSTRACT: DNA polymerase III mis-insertion may, where not corrected by its 3'→ 5' exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The k _cat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo⁸G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The K _M values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N ⁴- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

SUBMITTER: Tesfahun AN

PROVIDER: S-EPMC8278400 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein.

Tesfahun Almaz Nigatu AN Alexeeva Marina M Tomkuvienė Miglė M Arshad Aysha A Guragain Prashanna P Klungland Arne A Klimašauskas Saulius S Ruoff Peter P Bjelland Svein S

Frontiers in microbiology 20210630

DNA polymerase III mis-insertion may, where not corrected by its 3'→ 5' exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by ...[more]

PMID: 34276575

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Project description:Transcription factors recognize specific genomic sequences to regulate complex gene-expression programs. Although it is well-established that transcription factors bind to specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood1,2. Many DNA-binding proteins induce changes in the structure of the DNA outside the intrinsic B-DNA envelope. However, how the energetic cost that is associated with distorting the DNA contributes to recognition has proven difficult to study, because the distorted DNA exists in low abundance in the unbound ensemble3-9. Here we use a high-throughput assay that we term SaMBA (saturation mismatch-binding assay) to investigate the role of DNA conformational penalties in transcription factor-DNA recognition. In SaMBA, mismatched base pairs are introduced to pre-induce structural distortions in the DNA that are much larger than those induced by changes in the Watson-Crick sequence. Notably, approximately 10% of mismatches increased transcription factor binding, and for each of the 22 transcription factors that were examined, at least one mismatch was found that increased the binding affinity. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into 'super sites' that exhibit stronger affinity than any known canonical binding site. Determination of high-resolution X-ray structures, combined with nuclear magnetic resonance measurements and structural analyses, showed that many of the DNA mismatches that increase binding induce distortions that are similar to those induced by protein binding-thus prepaying some of the energetic cost incurred from deforming the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit transcription factors and thus modulate replication and repair activities in the cell10,11.

Dataset Information

Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein.

Publications

Alleviation of C⋅C Mismatches in DNA by the <i>Escherichia coli</i> Fpg Protein.

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