skip to main content

Explore Research Products in the NSF-PAR

Title: Transient kinetic analysis of oxidative dealkylation by the direct reversal DNA repair enzyme AlkB
AlkB is a bacterial Fe(II)– and 2-oxoglutarate–dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid–alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1, N 6 -ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3.  more » « less
Award ID(s):
1615586
NSF-PAR ID:
10156610
Author(s) / Creator(s):
; ;
Date Published:
Journal Name:
Journal of Biological Chemistry
Volume:
295
Issue:
21
ISSN:
0021-9258
Page Range / eLocation ID:
7317 to 7326
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ( , microPublication biology)
    The plant-specific RNA Polymerase V (Pol V) plays a key role in gene silencing, but its role in repair of double stranded DNA breaks is unclear. Excision of the transposable element mPing creates double stranded breaks that are repaired by NHEJ. We measured mPing excision site repair in multiple DNA methylation mutants including pol V using an mPing : GFP reporter. Two independent mutant alleles of pol V showed less GFP expression, indicating that the Pol V protein plays a role in excision site repair. Sequence analysis of the pol V excision sites indicated an elevated rate of large deletions consistent with less efficient repair. These results clarify the role of Pol V, but not other RNA-directed DNA methylation proteins (Pol IV) or maintenance DNA methylation pathways ( MET1 ), in the repair of double-strand DNA breaks. 
    more » « less
  2. ; ; ; ; ( , microPublication biology)
    The plant-specific RNA Polymerase V (Pol V) plays a key role in gene silencing, but its role in repair of double stranded DNA breaks is unclear. Excision of the transposable element mPing creates double stranded breaks that are repaired by NHEJ. We measured mPing excision site repair in multiple DNA methylation mutants including pol V using an mPing:GFP reporter. Two independent mutant alleles of pol V showed less GFP expression, indicating that the Pol V protein plays a role in excision site repair. Sequence analysis of the pol V excision sites indicated an elevated rate of large deletions consistent with less efficient repair. These results clarify the role of Pol V, but not other RNA-directed DNA methylation proteins (Pol IV) or maintenance DNA methylation pathways (MET1), in the repair of double-strand DNA breaks. 
    more » « less
  3. ; ; ( , Scientific Reports)
    Abstract

    Poly (ADP)-ribose polymerase 1 (PARP1) is an abundant nuclear protein well-known for its role in DNA repair yet also participates in DNA replication, transcription, and co-transcriptional splicing, where DNA is undamaged. Thus, binding to undamaged regions in DNA and RNA is likely a part of PARP1’s normal repertoire. Here we describe analyses of PARP1 binding to two short single-stranded DNAs, a single-stranded RNA, and a double stranded DNA. The investigations involved comparing the wild-type (WT) full-length enzyme with mutants lacking the catalytic domain (∆CAT) or zinc fingers 1 and 2 (∆Zn1∆Zn2). All three protein types exhibited monomeric characteristics in solution and formed saturated 2:1 complexes with single-stranded T20and U20oligonucleotides. These complexes formed without accumulation of 1:1 intermediates, a pattern suggestive of positive binding cooperativity. The retention of binding activities by ∆CAT and ∆Zn1∆Zn2 enzymes suggests that neither the catalytic domain nor zinc fingers 1 and 2 are indispensable for cooperative binding. In contrast, when a double stranded 19mer DNA was tested, WT PARP1 formed a 4:1 complex while the ∆Zn1Zn2 mutant binding saturated at 1:1 stoichiometry. These deviations from the 2:1 pattern observed with T20and U20oligonucleotides show that PARP’s binding mechanism can be influenced by the secondary structure of the nucleic acid. Our studies show that PARP1:nucleic acid interactions are strongly dependent on the nucleic acid type and properties, perhaps reflecting PARP1’s ability to respond differently to different nucleic acid ligands in cells. These findings lay a platform for understanding how the functionally versatile PARP1 recognizes diverse oligonucleotides within the realms of chromatin and RNA biology.

     
    more » « less
  4. ; ; ; ( , Physical Chemistry Chemical Physics)
    DNA alkylation is used as the key epigenetic mark in eukaryotes, however, most alkylation in DNA can result in deleterious effects. Therefore, this process needs to be tightly regulated. The enzymes of the AlkB and Ten-Eleven Translocation (TET) families are members of the Fe and alpha-ketoglutarate-dependent superfamily of enzymes that are tasked with dealkylating DNA and RNA in cells. Members of these families span all species and are an integral part of transcriptional regulation. While both families catalyze oxidative dealkylation of various bases, each has specific preference for alkylated base type as well as distinct catalytic mechanisms. This perspective aims to provide an overview of computational work carried out to investigate several members of these enzyme families including AlkB, ALKB Homolog 2, ALKB Homolog 3 and Ten-Eleven Translocate 2. Insights into structural details, mutagenesis studies, reaction path analysis, electronic structure features in the active site, and substrate preferences are presented and discussed. 
    more » « less
  5. ; ; ; ; ; ; ( , Nucleic Acids Research)
    Abstract Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (∼70–80 base pairs per second) unwinding extensive tracts (∼8–10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email