Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
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Dipivaloyl-5-carboxyfluorescein N-hydroxysuccinimidyl ester 1 and 5-propargylamino-2',3'-dideoxyuridine triphosphate 5 were modified with maleimide, haloacetamide, and sulfhydryl reactive functional groups to participate in cross-conjugation reactions via sulfide bonds to generate fluorescently labeled, thioether cross-conjugated terminators 10 and 11. Their DNA sequencing potential was compared with an amide cross-conjugated terminator 13, synthesized by directly coupling 5-carboxyfluorescein NHS ester with 18-ddUTP 9. These terminators (10, 11, and 13) in combination with the Thermo Sequenase II DNA polymerase, in thermal cycle sequencing experiments, revealed that the thioether cross-conjugated terminator 10 and amide cross-conjugated terminator 13 served as good terminating substrates, generating satisfactory single-color gel images and electropherograms, while the other thioether cross-conjugated and maleimide derived one 11 underwent unexpected pH and temperature induced decomposition without showing fluorescent signatures for incorporation.
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PMID:Utility of thiol-cross-linked fluorescent dye labeled terminators for DNA sequencing. 1200 35

Abasic (AP) sites are the most common lesions arising in genomic DNA. Repair of this potentially mutagenic DNA damage is initiated by the major apurinic/apyrimidinic endonuclease Ape1, which specifically recognizes and cleaves the DNA backbone 5' to the AP site. Ape1 is one of the major proteins in the base excision repair pathway (BER), and deletions in any of the BER proteins result in embryonic lethality. In this study, we employed fluorescence spectroscopy and in vitro mass spectrometric protein footprinting to investigate Ape1 conformational changes during various nucleoprotein interactions along its reaction pathway. Differences in intrinsic fluorescence emission spectra were observed during Ape1 protein's processing of the substrate, indicating possible conformational changes of the nucleoprotein complexes. To determine the protein domains that are involved in the putative conformational change, full-length Ape1 protein was probed with a lysine-reactive reagent (NHS-biotin) in the context of free protein and DNA-bound complexes. Protection patterns between pre- and postincision complexes revealed an increased susceptibility of lysine residues localized on the Ape1 surface that contacts the 3' end of the incised duplex (downstream of the incision site). We propose that the decreased protection results from Ape1 having a more relaxed grip on this section of the incised duplex to facilitate the handoff to the downstream BER enzyme. Protection of lysines (residues 24-35) in the N-terminal region was also observed in the intact AP-DNA-bound complex. These residues are part of the Ref1 domain which functions to regulate the activity of several transcription factors but to date has not been ascribed a DNA binding function. The reactivity of these Ref1 lysines was restored in the postincision complex. The differential protection patterns of lysines in the flexible N-terminal domain suggest a novel Ref1 conformational change concomitant with DNA binding and catalysis. It is likely that Ape1 employs this structural switch to mediate redox and nuclease activities. The ability of the Ape1-AP-DNA complex to recruit other BER proteins was also investigated by probing ternary complexes comprised of Ape1, DNA polymerase beta (Polbeta), and different BER DNA intermediates (abasic or gapped DNA). Our results suggest that Polbeta approaches the Ape1-DNA complex downstream of the incision site, displaces Ape1 DNA binding contacts (K227, K228, and K276), and in the process makes minimal interactions with lysine residues in the Ref1 domain.
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PMID:Probing conformational changes in Ape1 during the progression of base excision repair. 2037 4