DNA POLYMERASE
Updated 05-May-2020.
Mondo shtuff from around the internet, all about DNA POLYMERASE!
PDB101: Molecule of the Month: DNA Polymerase: DNA polymerase makes an accurate copy of the cell’s genome
Yeast DNA Polymerase ∊ Participates in Leading-Strand DNA Replication: Multiple DNA polymerases participate in replicating the leading and lagging strands of the eukaryotic nuclear genome. Although 50 years have passed since the first DNA polymerase was discovered, the identity of the major polymerase used for leading-strand replication is uncertain. We constructed a derivative of yeast DNA polymerase ∊ that retains high replication activity but has strongly reduced replication fidelity, particularly for thymine-deoxythymidine 5’-monophosphate (T-dTMP) but not adenine-deoxyadenosine 5’-monophosphate (A-dAMP) mismatches. Yeast strains with this DNA polymerase ∊ allele have elevated rates of T to A substitution mutations. The position and rate of these substitutions depend on the orientation of the mutational reporter and its location relative to origins of DNA replication and reveal a pattern indicating that DNA polymerase ∊ participates in leading-strand DNA replication.
Primary Structure of the Catalytic Subunit of Human DNA Polymerase δ and Chromosomal Location of the Gene: The catalytic subunit (Mr approximately 124,000) of human DNA polymerase delta has been cloned by PCR using poly(A)+ RNA from HepG2 cells and primers designed from the amino acid sequence of regions highly conserved between bovine and yeast DNA polymerase delta. The human cDNA was 3443 nucleotides in length and coded for a polypeptide of 1107 amino acids. The enzyme was 94% identical to bovine DNA polymerase delta and contained the numerous highly conserved regions previously observed in the bovine and yeast enzymes. The human enzyme also contained two putative zinc-finger domains in the carboxyl end of the molecule, as well as a putative nuclear localization signal at the amino-terminal end. The gene coding for human DNA polymerase delta was localized to chromosome 19.
Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography: Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same `double-psi β-barrel’ architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs.
Managing DNA polymerases: Coordinating DNA replication, DNA repair, and DNA recombination: Two important and timely questions with respect to DNA replication, DNA recombination, and DNA repair are: (i) what controls which DNA polymerase gains access to a particular primer-terminus, and (ii) what determines whether a DNA polymerase hands off its DNA substrate to either a different DNA polymerase or to a different protein(s) for the completion of the specific biological process? These questions have taken on added importance in light of the fact that the number of known template-dependent DNA polymerases in both eukaryotes and in prokaryotes has grown tremendously in the past two years. Most notably, the current list now includes a completely new family of enzymes that are capable of replicating imperfect DNA templates. This UmuC-DinB-Rad30-Rev1 superfamily of DNA polymerases has members in all three kingdoms of life. Members of this family have recently received a great deal of attention due to the roles they play in translesion DNA synthesis (TLS), the potentially mutagenic replication over DNA lesions that act as potent blocks to continued replication catalyzed by replicative DNA polymerases. Here, we have attempted to summarize our current understanding of the regulation of action of DNA polymerases with respect to their roles in DNA replication, TLS, DNA repair, DNA recombination, and cell cycle progression. In particular, we discuss these issues in the context of the Gram-negative bacterium, Escherichia coli, that contains a DNA polymerase (Pol V) known to participate in most, if not all, of these processes.
Single-Molecule DNA Polymerase Dynamics at a Bacterial Replisome in Live Cells
DNA Polymerase Families: The DNA polymerases are divided into seven families based on their sequence homology and crystal structure analysis. These include families A, B, C, D, X, Y and RT.
My botty best at summarizing from Wikipedia: DNA polymerase is an enzyme that synthesizes DNA molecules from deoxyribonucleotides, the building blocks of DNA . enzymes are essential for DNA replication and usually work in pairs to create two identical DNA before replication can take place, an enzyme unwinds the DNA molecule from its tightly woven form . this opens up or “unzips” the double-stranded DNA to give two single strands of DNA DNA polymerase II was also discovered by Thomas Kornberg and Malcolm E. Gefter in 1970 . RNA polymerases synthesize RNA from ribonucleotides from either RNA or no known DNA polymerase is able to begin a new chain (de novo) it can only add a nucleotide onto a pre-existing 3′-OH group . primers consist of DNA polymerase requires a free 3′ OH group for initiation of synthesis . it can synthesize in only one direction by extending the 3′ end of the preexisting nucleotide chain error correction is a property of some, but not all DNA polymerases . the 3’–5′ exonuclease activity of the enzyme allows the incorrect base pair to be excised . after base mismatches in DNA base pairing can potentially result in dysfunctional proteins and could lead to cancer . many DNA polymerases contain an exonuclease domain . this acts in detecting base pair mismatch hydrogen bonds play a key role in base pair binding and interaction . loss of interaction said to trigger shift in balance for binding template-primer . incorporation of wrong nucleotide causes retard in DNA polymerization in a purine:pyrimidine mismatch there is a displacement of the purine towards the major groove . steric clashes occur and important van der Waals and electrostatic interactions are lost . DNA poly the shape can be described as resembling a right hand with thumb, finger, and palm domains . palm domain appears to function in catalyzing the transfer of phosphoryl groups . DNA is bound to thumb domain plays a potential role in the processivity, translocation, and positioning of the DNA . processivity is a characteristic of enzymes that function on polymeric substrates . average DNA polymerase requires about one second processive DNA polymerases add multiple nucleotides per second . rate of DNA synthesis was determined by rate of phage T4 DNA elongation . DNA polymerase’s ability to slide there is a dramatic increase in processivity at the replication fork . this increase is facilitated by the DNA polymerase’s association with the sliding DNA clamp . the clamps are multiple protein subunits associated in the retroviruses encode an unusual DNA polymerase called reverse transcriptase . it polymerizes DNA from a template of RNA . some viruses also encode special DNA polymerases . core polymerase synthesizes DNA from the DNA template but it cannot initiate the synthesis alone or accurately . holoenzyme accurately initiates synthesis . Pol I starts adding nucleotides at the RNA primer:template junction known as the origin of replication (ori) Pol III holoenzyme is assembled and takes over replication at a highly processive Detailed classification divides family B in archaea into B1, B2, B3 . some viruses and mitochondrial plasmids carry polB as well . it consists of three assemblies: the pol family C polymerases are a subcategory of Family X with no eukaryotic equivalents . the old textbook “trombone model” depicts an elongation complex with two equivalents leading strand synthesis may not be completely continuous . pol III* has a high frequency of dissociation from active RFs . replication fork turnover rate was about 10s for Pol III*, 47s Pol IV is a Family Y polymerase expressed by the dinB gene . it is switched on via SOS induction caused by stalled polymerases at the replication fork . During SOS Stalled polymerases causes RecA to bind to ssDNA, which causes LexA protein to autodigest . LexA then loses ability to repress transcription of umuDC operon . the involvement of more than one TLS polymerase working in succession to bypass a lesion has not yet been shown in E. coli . pol IV can catalyze both insertion and extension with high efficiency the DP1-DP2 interface resembles that of Eukaryotic Class B polymerase zinc finger . DP1, a Mre11-like exonuclease, is likely the precursor of Pyrococcus abyssi polD is more heat-stable and more accurate than Taq polymerase . family X polymerases are found mainly in vertebrates, and a Pol and pol are involved in non-homologous end-joining . Pol is expressed by genes POLD1, POLD2, POLD3 and POLD4 . TdT Pol is encoded by the POLE1, the catalytic subunit, POLE2, and POLE3 gene . pol ‘s C-terminus “polymerase relic” region zinc finger has implications in the origins of Eukaryota, which is placed into the Asgard group with archaeal B3 polymerase . members of Family Y have five common motifs to aid in Pol is thought to act as an extender or an inserter of a specific base at certain DNA lesions . researchers have found two probable functions of pol . there are two pathways of damage repair leading Pol lacks 3′ to 5’ exonuclease activity, is unique in that it can extend primers with terminal mismatches . Rev1 has three regions of interest in the BRCT loss of REV3 gene in budding yeast can cause increased sensitivity to DNA-damaging agents . collapse of replication forks where replication polymerases have stalled . gradual decrease in size of telome any mutation that leads to limited or non-functioning Pol has a significant effect on mtDNA and is the most common cause of autosomal inherited mitochondrial disorders . pol contains a many homologs of Pol , encoded by the POLQ gene, are found in eukaryotes . but its function is not clearly understood . the sequence of amino acids in the C-termin DNA polymerase nu plays an active role in homology repair during cellular responses to crosslinks . plants use two Family A polymerases to copy both the mitochrondrial and plastid genomes