Recognition of the Pro-mutagenic Base Uracil by Family B DNA Polymerases from Archaea

Shuttleworth, G. R.; Fogg, Mark J.; Kurpiewski, Michael R.; Jen‐Jacobson, Linda; Connolly, Bernard A.

doi:10.1016/j.jmb.2004.01.021

Cited by 38 publications

(89 citation statements)

References 52 publications

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“…and other archaea possess only family-B polymerases capable of genome replication (Kawarabayasi et al 2001;She et al 2001). Archaeal family-B polymerases have the peculiar characteristic of stalling just before template deoxyuridine residues (Lasken et al 1996;Shuttleworth et al 2004) although, to our knowledge, the functional significance of this property has not been confirmed in vivo. T. thermophilus and S. acidocaldarius have different types of DNA-binding proteins (Zierer and Choli 1990;Du and Pène 1999) and different numbers of replication origins (Lundgren et al 2003;Henne et al 2004).…”

Section: Discussionmentioning

confidence: 91%

The Rate and Character of Spontaneous Mutation in Thermus thermophilus

et al. 2008

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Selection of spontaneous, loss-of-function mutations at two chromosomal loci (pyrF and pyrE) enabled the first molecular-level analysis of replication fidelity in the extremely thermophilic bacterium Thermus thermophilus. Two different methods yielded similar mutation rates, and mutational spectra determined by sequencing of independent mutants revealed a variety of replication errors distributed throughout the target genes. The genomic mutation rate estimated from these targets, 0.00097 6 0.00052 per replication, was lower than corresponding estimates from mesophilic microorganisms, primarily because of a low rate of base substitution. However, both the rate and spectrum of spontaneous mutations in T. thermophilus resembled those of the thermoacidophilic archaeon Sulfolobus acidocaldarius, despite important molecular differences between these two thermophiles and their genomes.

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Section: Discussionmentioning

confidence: 91%

The Rate and Character of Spontaneous Mutation in Thermus thermophilus

et al. 2008

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“…8B). We mutated Val 17 to a glutamate, which we predicted would flip out into the solvent, destroying the pocket in a manner similar to that seen recently with a DNA polymerase (38). We also mutated Gly 18 to an aspartate, which would also destroy the pocket.…”

Section: The Isolated Wedge Domain Binds a Holliday Junction-mentioning

confidence: 97%

DNA Binding by the Substrate Specificity (Wedge) Domain of RecG Helicase Suggests a Role in Processivity

Briggs¹,

Mahdi²,

Qin

et al. 2005

Journal of Biological Chemistry

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RecG differs from most helicases acting on branched DNA in that it is thought to catalyze unwinding via translocation of a monomer on dsDNA, with a wedge domain facilitating strand separation. Conserved phenylalanines in the wedge are shown to be critical for DNA binding. When detached from the helicase domains, the wedge bound a Holliday junction with high affinity but failed to bind a replication fork structure. Further stabilizing contacts are identified in full-length RecG, which may explain fork binding. Detached from the wedge, the helicase region unwound junctions but had extremely low substrate affinity, arguing against the "classical inchworm" mode of translocation. We propose that the processivity of RecG on branched DNA substrates is dependent on the ability of the wedge to establish strong binding at the branch point. This keeps the helicase motor in contact with the substrate, enabling it to drive dsDNA translocation with high efficiency.Helicases are ubiquitous enzymes essential in all stages of DNA and RNA metabolism, including replication, transcription, recombination, and repair (1, 2). They are motor proteins that generally translocate along one or both strands of dsDNA, 1 dsRNA, or a DNA-RNA hybrid in an ATP-dependent manner and separate some or all parts of the molecule into its component strands. Some enzymes are highly processive, driving strand separation for long distances without dissociating from the template, a property that is especially important for enzymes involved in chromosome replication (3).A variety of mechanisms has evolved to ensure processivity. The prokaryotic helicase DnaB, for example, forms a hexameric ring that completely encircles the DNA, which allows onedimensional motion while preventing dissociation (4, 5). Such a strategy is common to many of the hexameric helicases (6). Other helicases are thought to achieve the same effect by interacting with the "sliding clamp" that encircles the DNA strands during replication. Examples include Wrn and Rrm3, which have been shown to have enhanced processivity in the presence of PCNA (7,8). The RecC component of the RecBCD helicase has no helicase activity in itself, but its role may be similar to a sliding clamp, preventing dissociation of the RecBCD complex from the DNA (9 -11).A second strategy is for the helicase to have two binding sites, such that one site can release and move along the strand while the second stays bound. This can be achieved by having a dimeric structure, with one binding site per monomer and has led to the proposal of the "hand over hand" or "rolling" method. Rep helicase is thought to translocate using this mechanism (12). The Rep dimer binds to DNA, with one monomer behind the other. ATP hydrolysis alters the conformation of the helicase, radically changing the position of one monomer relative to the other, as well as causing a change in its nucleic acid binding properties (12)(13)(14). This movement means the second monomer can now bind the DNA in front of the first monomer, and thus translocation i...

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“…Given the ability of the 3D-PCR to differentially amplify HIV-1 G 3 A hypermutants, we reasoned that despite residual plasmid DNA from transfection, the method should be able to selectively amplify de novo synthesized and hyperdeaminated HBV DNA from a total DNA extraction. As hyperdeaminated DNA contains multiple uracil residues, Taq polymerase was used, as opposed to Pfu (20), because the latter has a read-ahead function and stalls at uracil residues in template DNA (28,29).…”

Section: Apobec3 Proteinsmentioning

confidence: 99%

Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo

Suspène

Guétard

Mathieu

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

280

266

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Because the replication of hepatitis B virus (HBV) proceeds via an obligatory reverse transcription step in the viral capsid, cDNA is potentially vulnerable to editing by cytidine deaminases of the APOBEC3 family. To date only two edited HBV genomes, referred to as G 3 A hypermutants, have been described in vivo. Recent work suggested that HBV replication was indeed restricted by APOBEC3G but by a mechanism other than editing. The issue of restriction has been explored by using a sensitive PCR method allowing differential amplification of AT-rich DNA. G 3 A hypermutated HBV genomes were recovered from transfection experiments involving APOBEC3B, -3C, -3F, and -3G indicating that all four enzymes were able to extensively deaminate cytidine residues in minus-strand DNA. Unexpectedly, three of the four enzymes (APOBEC3B, -3F, and -3G) deaminated HBV plus-strand DNA as well. From the serum of two of four patients with high viremia, G 3 A hypermutated genomes were recovered at a frequency of Ϸ10 ؊4 , indicating that they are, albeit relatively rare, part of the natural cycle of HBV infection. These findings suggest that human APOBEC3 enzymes can impact HBV replication via cytidine deamination.hypermutation ͉ DNA editing G 3 A hypermutated retroviral genomes result from the editing of nascent DNA by APOBEC3 cytidine deaminases (1-7). This was originally demonstrated for HIV-1 and the APOBEC3G member of a cluster of seven genes (3A-3H) on human chromosome 22 (8, 9). Editing occurs on the background of a ⌬vif genome (1-5, 10). The HIV Vif protein prevents packaging of either APOBEC3F or -3G resulting in their ubiquitination and degradation by the proteasomal pathway (11)(12)(13)(14). Editing of cytidine residues in neo-synthesized minusstrand cDNA results in the formation of multiple uracil residues that are read as T during plus-strand synthesis. Albeit referred to as G 3 A hypermutants by reference to the viral plus strand, mechanistically the action is occurring on the minus strand and independently of reverse transcriptase (7). Up to 60% of G residues in a lentiviral genome can be substituted resulting in the total loss of information (15-17). As such, APOBEC3F and -3G constitute a powerful restriction mechanism to reverse transcription. Retroviruses have either to avoid replication in cells expressing APOBEC3 molecules or else evolve a mechanism that neutralizes their effect. The vif gene of human and primate lentiviruses, as well as their homologues in most of the other lentiviruses, reflect the latter solution.Hepatitis B viruses (HBVs) replicate via an obligate reverse transcription step occurring in a capsid structure close to the endoplasmic reticulum. A pair of G 3 A hypermutated genomes were identified in the blood of a chronically infected patient yet have remained unique despite a burgeoning database (18). Recent reports demonstrated that HBV replication could be strongly restricted by APOBEC3G and -3F in an experimental setting (20,21). Restriction was highlighted by a strong reduction in the proportion o...

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Recognition of the Pro-mutagenic Base Uracil by Family B DNA Polymerases from Archaea

Cited by 38 publications

References 52 publications

The Rate and Character of Spontaneous Mutation in Thermus thermophilus

The Rate and Character of Spontaneous Mutation in Thermus thermophilus

DNA Binding by the Substrate Specificity (Wedge) Domain of RecG Helicase Suggests a Role in Processivity

Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo

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