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Add multiple products. Please Enquire This product is discontinued. Add to Helix This product is available through the Promega Helix onsite stocking program. DNA Polymerase I. Applications Labeling of DNA to high specific radioactivity by nick translation. Second-strand cDNA synthesis.
References Kelly, R. Harwood, S. Source: Recombinant E. You are viewing: M Change Configuration. Certificate of Analysis Search by lot number. Not for Use in Diagnostic Procedures.
Storage Conditions. Citations A novel highly divergent strain of cell fusing agent virus CFAV in mosquitoes from the Brazilian amazon region. Let's find the product that meets your needs.
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In addition, note how the template G that base pairs with the ddCTP in the closed conformation, moves away from the active site in the open conformation, in which it has no base pairing partner.
A closeup of the active site region in the right reveals that the side chain of the conserved Tyr colored with C pink is stacked on top of the template G that forms a base pair with the bound ddCTP, where it apparently participates in verifying that a Watson—Crick base pair has formed.
In the left , Tyr , which is part of the fingers domain, has moved aside, presumably to permit the active site to form about the incoming dNTP satisfy yourself that the Tyr side chain is stacked on the template G in the open form but not in the closed form. Jump to: navigation , search. Show: Asymmetric Unit Biological Assembly. Export Animated Image. Pol I has three active sites: 1. Klentaq1—Closed conformation 3ktq. Klentaq1—Open conformation 2ktq. DNA polymerase IV and V have large active sites that allow for more base misincorporation, and are therefore more error-prone.
They also lack proofreading-exonuclease subunits to correct misincorporations Nohmi , and Hastings et al. The palm region is thought to catalyze the phosphoryl transfer, and the finger region is thought to interact with the incoming nucleoside triphosphate and the template base it is paired to.
The gene encoding DNA polymerase I polA contains approximately 3, base pairs and encodes approximately 1, amino acid residues in a simple polypeptide chain. Even organisms separated by a billion years of evolution such as Deinococcus-Thermus genera and E. Place Order. This strongly argues against local sequence context hotspot effects on the fidelity of pol I synthesis; instead it is more consistent with a change in template altering the spectrum of mutations without changing its overall frequency.
Thus, overall the mutation profile of the lagging-strand mutation clusters identified in Figure 5 suggests that these clusters most likely represent OP sites. The sequence is grayed out and mutant positions are highlighted in black. We also indicate the estimated length in nucleotides of the site, as well as the distance to the next marker lagging-strand mutation.
To facilitate visualizing these sequences in a wider sequence context, we also highlighted putative OP sites in Supplementary Figure S1. Okazaki processing site sequence context. Mutant positions are highlighted in bold black on a grayed out sequence. Sequence intervals defined by marker lagging-strand mutation clusters Figure 5 are highlighted light gray box and the total number of nucleotides within each interval is listed. Arrows represent the distance in nucleotides, listed on top from the Okazaki processing site to the next lagging-strand mutation marker.
Thus, the sequence context of lagging-strand mutation clusters is also consistent with their interpretation as OP sites. DNA polymerase I is one of five known polymerases expressed by E. Even though pol I was the first polymerase to be discovered, some questions regarding its function in vivo remain. Here, we address some of these questions by using the footprint of pol I—generated mutations in neutral sequence to define pol I replication templates.
We increased the mutation frequency of pol I by genetically altering the fidelity of this polymerase. Low fidelity pol I mutations may admittedly have pleiotropic effects such as altering the processivity of the polymerase, its efficiency to exchange with other polymerases, or its nick-translation activity. However, the dramatic increase in mutation frequency produced by our low-fidelity polymerase was critical for our experimental approach for two reasons:.
Minimal background from other mutation sources: the mutation rate of the muta-plasmid system is so far above that of spontaneous mutagenesis that it virtually guarantees that all mutations sequenced are produced by pol I. There is no question about the source of mutations in our system because the frequency of ColE1 plasmid mutation in vivo correlates directly with the fidelity of individual error-prone pol I alleles expressed Direct sequencing: our elevated mutation frequency allowed efficient data collection by direct sequencing, bypassing the need for reporters.
The absence of any significant functional selection allowed the generation of an accurate spectrum of mutations in vivo , and even more importantly of accurate data on the physical distribution of mutations along the plasmid sequence. We have recently reported a very similar decrease in mutation frequency with increasing distance from ColE1 ori using a streamlined muta-plasmid mutagenesis protocol This mutation frequency profile is consistent with a switch to pol III replication, as pol III is a high-fidelity polymerase and therefore not expected to leave a detectable mutation footprint in our system.
This gradual transition to pol III replication may be a default mechanism in the absence of the ssiB primosome assembly site for the leading strand 25 , 26 ; alternatively, in ColE1 plasmids the switch between pol I and pol III polymerases may be far more gradual than previously thought. Given that we sequenced the leader strand, we can assume that the mutation spectrum in proximal areas of the plasmid shown in Figure 4 a approximates the error rate of the polymerase in vivo , after proofreading and mismatch repair Figure 3 a, Scenario 1.
The contribution of mismatch repair, which preferentially resolves frameshifts and transitions 34 to the error rates of pol I in vivo , is unclear. The polA12 strain we used as a host is mismatch repair-proficient. Widespread mutagenesis can saturate mismatch repair In our case, mutagenesis is largely targeted to ColE1 plasmid sequences 15 and is therefore less likely to saturate the mismatch repair capacity of the cell than non-targeted in vivo mutagenesis.
The asymmetry between complementary mutations mentioned above can be interpreted as a difference in the error rate of the polymerase for the two complementary mutations in vivo Figure 3 a, Scenario 1. It agrees, however, with another study of ColE1 plasmid replication in vivo showing that inhibition of primosome assembly through a dnaT mutation or by treatment with anti-dnaT antibodies results in 0.
This inversion suggests that leading-strand synthesis in this area is negligible Figure 3 a, Scenario 2. This observation agrees with reports showing that pol III is essential for completion of ColE1 plasmid replication 38 and supports our use of marker mutations to identify strand preferences in replication.
Next, we looked for a mutation footprint that may correspond to Okazaki primer processing. We reasoned that mutations in proximal areas that show a negative bias in frequency compared to their complementary ones Figure 4 a should be enriched in areas of lagging-strand synthesis Figure 3.
We confirmed this approach showing that these marker mutations are enriched at distal positions Figure 4 c. The distribution and spectrum of mutations at these sites argues strongly against clustering due to a local increase in polymerase error associated with sequence context hotspot effects. We can distinguish four lines of evidence supporting our proposition that instead lagging-strand mutation clusters represent OP sites.
Inverted bias: marker lagging-strand mutations are overrepresented and their complementary mutations underrepresented at these sites Figure 6. Strikingly, the enrichment for marker lagging-strand mutations was not limited to one or two types; instead we found that all six types of marker point mutations were enriched, between 2. The most parsimonious explanation in this comprehensive shift in mutation pattern is a template switch. No evidence for local increase in error rate: a local increase in polymerase error rate typically results in multiple hits in one or a few two to three adjacent positions, such as we saw in the hTK hotspots Supplementary Figure S2 or as previously reported for the lacI reporter gene We found this motif at the expected location in all OP sites but one highlighted in Figure 7.
Therefore each iterative cycle of mutagenesis should increase the differential between frequencies of leading- versus lagging-strand replication. This prediction agrees with our experimental data: for comparable numbers of lagging-strand mutations 97 versus 80 we see a much higher number mutations per site in the hTK library relative to the pGFPuv library 8.
While we used proximity between lagging-strand mutations to identify these sites, we did not assume any particular size. That previously reported work was done in a rnhA strain of E.
The specific location of the OP sites varies between the two libraries, with the exception of the site closest to the ssiA site at positions — Figure 5 , which is in an area of sequence shared between the two libraries. This suggests that primase recognition is sequence-context dependent. The preferred primase recognition motif is on the lagging strand GTC 28 , 39 , We ignore whether ATT TAA on the leading-strand sequence represents a preferred primase motif in ColE1 plasmids or whether this is a serendipitous finding due to the small number of OP sites represented in our study.
In sum, based on at least five different criteria regarding mutation distribution and spectrum, positional enrichment, size and flanking sequence context we have identified a mutational footprint very likely corresponding to lagging-strand processing by pol I.
Given that the different modalities of pol I replication in the cell differ mostly at the initiation steps, our observations regarding the transition between pol I and pol III replication and Okazaki fragment processing likely apply to pol I genomic replication and DNA repair as well. The primosome assembled for initiation of ColE1 plasmid replication is essentially identical to the PriA-dependent replisome recruited to R- or D-loops during DNA repair 6 , Gaps that form during lagging-strand synthesis are known to play a major role in processing replication blocks by facilitating strand-switch and replication fork reversal 43— If confirmed, the presence of short Okazaki fragments during PriA-dependent replication could assist in the processing of replication blocks.
Mutational footprinting has allowed us to follow the switch from pol I to pol III replication during leading-strand synthesis and even RNA processing by pol I on the lagging strand, which is restricted to very short sequences. Finally, our mutational footprinting approach can also be used more broadly to study processing of specific lesions by individual polymerases in vivo and to investigate how polymerase activity may be affected by sequence topology or by interactions with protein partners such as DNA repair or processivity factors.
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