Wardle J, Burgers P M, Cann I K, et al. Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya. Nucleic Acids Res, 2008

Institute for Cell and Molecular Biosciences (ICaMB), University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
Nucleic Acids Research (Impact Factor: 9.11). 03/2008; 36(3):705-11. DOI: 10.1093/nar/gkm1023
Source: PubMed


Family B DNA polymerases from archaea such as Pyrococcus furiosus, which live at temperatures approximately 100 degrees C, specifically recognize uracil in DNA templates and stall replication in response to this base. Here it is demonstrated that interaction with uracil is not restricted to hyperthermophilic archaea and that the polymerase from mesophilic Methanosarcina acetivorans shows identical behaviour. The family B DNA polymerases replicate the genomes of archaea, one of the three fundamental domains of life. This publication further shows that the DNA replicating polymerases from the other two domains, bacteria (polymerase III) and eukaryotes (polymerases delta and epsilon for nuclear DNA and polymerase gamma for mitochondrial) are also unable to recognize uracil. Uracil occurs in DNA as a result of deamination of cytosine, either in G:C base-pairs or, more rapidly, in single stranded regions produced, for example, during replication. The resulting G:U mis-pairs/single stranded uracils are promutagenic and, unless repaired, give rise to G:C to A:T transitions in 50% of the progeny. The confinement of uracil recognition to polymerases of the archaeal domain is discussed in terms of the DNA repair pathways necessary for the elimination of uracil.

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    • "Using these methods, only the PCR products that contain uracil are enzymatically digested; therefore, any contaminating PCR products can be digested with no risk of destroying the target DNA about to be amplified. Unfortunately, uracilated DNA is not amplified well with widely-used emulsion or cluster PCR kits, due to the use of uracil-illiterate polymerases in most next generation sequencing platforms [2]. "
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    • "The catalytic core of yeast Pol d aligns with RB69 DNA Pol in the general plan of organization with unique structural features [Swan et al., 2009]. All Pols possess remnants of the uracil recognizing domain but do not sense uracil like their archael homologs [Wardle et al., 2008]. The C-terminal domain of Pols (CTD) has two cysteine-rich metal binding motifs (MBM1 and MBM2) critical for the assembly of the holoenzymes [Dua et al., 1998; Sanchez Garcia et al., 2004; Klinge et al., 2009; Tahirov et al., 2009]. "
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    • "Another case of functional inactivation despite structural conservation is the uracil recognition domain that is conserved in archaeal and eukaryotic B-family polymerases (Fig 4) but lost the capacity to sense uracil in front of the moving polymerase in eukaryotes [50]. Mechanistic characterization of the inactivated polymerase subunits and domains is expected to shed new light on the functions of the replication apparatus. "
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    ABSTRACT: Evolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved. We performed a comparative analysis of archaeal, eukaryotic, and bacterial B-family DNA polymerases, which are the main replicative polymerases in archaea and eukaryotes, combined with an analysis of domain architectures. Surprisingly, we found that eukaryotic Polymerase epsilon consists of two tandem exonuclease-polymerase modules, the active N-terminal module and a C-terminal module in which both enzymatic domains are inactivated. The two modules are only distantly related to each other, an observation that suggests the possibility that Pol epsilon evolved as a result of insertion and subsequent inactivation of a distinct polymerase, possibly, of bacterial descent, upstream of the C-terminal Zn-fingers, rather than by tandem duplication. The presence of an inactivated exonuclease-polymerase module in Pol epsilon parallels a similar inactivation of both enzymatic domains in a distinct family of archaeal B-family polymerases. The results of phylogenetic analysis indicate that eukaryotic B-family polymerases, most likely, originate from two distantly related archaeal B-family polymerases, one form giving rise to Pol epsilon, and the other one to the common ancestor of Pol alpha, Pol delta, and Pol zeta. The C-terminal Zn-fingers that are present in all eukaryotic B-family polymerases, unexpectedly, are homologous to the Zn-finger of archaeal D-family DNA polymerases that are otherwise unrelated to the B family. The Zn-finger of Polepsilon shows a markedly greater similarity to the counterpart in archaeal PolD than the Zn-fingers of other eukaryotic B-family polymerases. Evolution of eukaryotic DNA polymerases seems to have involved previously unnoticed complex events. We hypothesize that the archaeal ancestor of eukaryotes encoded three DNA polymerases, namely, two distinct B-family polymerases and a D-family polymerase all of which contributed to the evolution of the eukaryotic replication machinery. The Zn-finger might have been acquired from PolD by the B-family form that gave rise to Pol epsilon prior to or in the course of eukaryogenesis, and subsequently, was captured by the ancestor of the other B-family eukaryotic polymerases. The inactivated polymerase-exonuclease module of Pol epsilon might have evolved by fusion with a distinct polymerase, rather than by duplication of the active module of Pol epsilon, and is likely to play an important role in the assembly of eukaryotic replication and repair complexes.
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