Abstract：
:HAP1, also known as APE/Ref-1, is the major apurinic/apyrimidinic (AP) endonuclease in human cells. Previous structural studies have suggested a possible role for the Asp-210 residue of HAP1 in the enzymatic function of this enzyme. Here, we demonstrate that substitution of Asp-210 by Asn or Ala eliminates the AP endonuclease activity of HAP1, while substitution by Glu reduces specific activity approximately 500-fold. Nevertheless, these mutant proteins still bind efficiently to oligonucleotides containing either AP sites or the chemically unrelated bulky p-benzoquinone (pBQ) derivatives of dC, dA and dG, all of which are substrates for HAP1. These results indicate that Asp-210 is required for catalysis, but not substrate recognition, consistent with enzyme kinetic data indicating that the HAP1-D210E protein has a 3000-fold reduced K(cat )for AP site cleavage, but an unchanged K(m). Through analysis of the binding of Asp-210 substitution mutants to oligonucleotides containing either an AP site or a pBQ adduct, we conclude that the absence of Asp-210 allows the formation of a stable HAP1-substrate complex that exists only transiently during the catalytic cycle of wild-type HAP1 protein. We interpret these data in the context of the structure of the HAP1 active site and the recently determined co-crystal structure of HAP1 bound to DNA substrates.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Rothwell DG,Hang B,Gorman MA,Freemont PS,Singer B,Hickson IDdoi
10.1093/nar/28.11.2207keywords:
subject
Has Abstractpub_date
2000-06-01 00:00:00pages
2207-13issue
11eissn
0305-1048issn
1362-4962journal_volume
28pub_type
杂志文章abstract：:Single-stranded guanosine-rich oligodeoxyribonucleotides (GROs) have a propensity to form quadruplex structures that are stabilized by G-quartets. In addition to intense speculation about the role of G-quartet formation in vivo, there is considerable interest in the therapeutic potential of quadruplex oligonucleotides...
journal_title：Nucleic acids research
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pub_type： 杂志文章
doi：10.1093/nar/gkl850
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journal_title：Nucleic acids research
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更新日期：1997-05-15 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/23.23.4864
更新日期：1995-12-11 00:00:00
abstract：:In order to further elucidate the mechanism of tRNA methylase-tRNA intreaction the methylation of some individual tRNAs separately and by pairs was performed. In conditions of tRNA excess the methylation rates of positionally analogous nucleotides in tRNA molecules are not summed up when two substrates are simultaneou...
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pub_type： 杂志文章
doi：10.1093/nar/3.8.2079
更新日期：1976-08-01 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/12.15.6031
更新日期：1984-08-10 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章
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更新日期：1992-02-11 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkq1034
更新日期：2011-03-01 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章,评审
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更新日期：2019-04-23 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/19.8.1759
更新日期：1991-04-25 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkt233
更新日期：2013-05-01 00:00:00
abstract：:The shape of the chromosomal DNA of the sulfur-dependent archaebacterium Sulfolobus acidocaldarius was analyzed by the pulsed-field gel electrophoresis(PFGE). S.acidocaldarius DNA digested with Notl showed two DNA bands at around 1.0 Mbp and 2.1 Mbp. Notl-linking clones were isolated from the library of S.acidocaldari...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/18.5.1133
更新日期：1990-03-11 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkz860
更新日期：2020-01-08 00:00:00
abstract：:Multiplexed tandem PCR (MT-PCR) is a process for highly multiplexed gene expression profiling. In the first step, multiple primer pairs are added to the RNA to be analysed together with reverse transcriptase and Taq DNA polymerase. Following reverse transcription, the multiplexed amplicons are simultaneously amplified...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gni182
更新日期：2005-11-24 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/19.11.3047
更新日期：1991-06-11 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkn292
更新日期：2008-07-01 00:00:00
abstract：:A-tracts are functionally important DNA sequences which induce helix bending and have peculiar structural properties. While A-tract structure has been qualitatively well characterized, their mechanical properties remain controversial. A-tracts appear structurally rigid and resist nucleosome formation, but seem flexibl...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gku338
更新日期：2014-06-01 00:00:00
abstract：:Pig Genomic Information System (PigGIS) is a web-based depository of pig (Sus scrofa) genomic learning mainly engineered for biomedical research to locate pig genes from their human homologs and position single nucleotide polymorphisms (SNPs) in different pig populations. It utilizes a variety of sequence data, includ...
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更新日期：2007-01-01 00:00:00
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更新日期：1984-04-25 00:00:00
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更新日期：2003-07-15 00:00:00
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更新日期：1974-02-01 00:00:00
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更新日期：2012-12-01 00:00:00
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pub_type： 杂志文章
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更新日期：2003-11-15 00:00:00
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更新日期：2015-02-18 00:00:00
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pub_type： 杂志文章
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更新日期：1993-12-25 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章
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更新日期：2012-03-01 00:00:00
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journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkm889
更新日期：2008-01-01 00:00:00
