helicase exonuclease in dna replication

... points 23 FIGURE 2.1 DNA replication licensing control by Geminin and CDKs 42 during the cell cycle FIGURE 2.2 Geminin Binding Domain of Human Cdt1 and Cdt1 44 Binding Domain of Human Geminin ... CDKs Geminin (Licensing) Figure2.1 DNA replication licensing control by Geminin and CDKs during the cell cycle In metazoa, Geminin, present from the onset of S phase to the end of M phase, binds ... information will prove useful in elucidating how Cdt1 and Geminin interact at the protein level We identified, cloned and expressed (in bacterial cells) the geminin-binding domain of human Cdt1 and purified...

Ngày tải lên: 04/10/2015, 10:24

... domains of both proteins are shown in cyan with ADP indicated in red The helix-turn-helix (HTH)-containing DNA binding domain of DnaA is in green and the winged helix (WH)-containing domain of ... Cdc6 proteins [60,61] As can be seen in Fig 4, both proteins possess N-terminal AAA+ domains and C-terminal DNA binding domains (DBDs) In DnaA, the DBD contains a helix-turn-helix; in the archaeal ... affinity of DnaC for single stranded DNA In contrast ADP– DnaC does not inhibit DnaB helicase and has lower DNA binding affinity Thus, it has been proposed that ATP–DnaC interacts with DnaB and facilitates...

Ngày tải lên: 07/03/2014, 21:20

... absolutely inactive in initiating SV40 DNA replication in an assay consisting of purified enzymes [26,27] We then reproduced the absence of JCV species specificity using the monopolymerase DNA replication ... facilitate initiation of replication in vivo but not in vitro, perhaps by alleviating nucleosome repression or by assisting in origin unwinding under conditions of altered template DNA superhelicity ... SV40 TAg and DNA with the SV40 replication origin (A); the JCV system contains JCV TAg and DNA with the JCV replication origin (B) DNA polymerase (0.25 and 0.5 U) of the indicated DNA polymerase...

Ngày tải lên: 17/03/2014, 03:20

... be within the interior region of a co-infecting T4 DNA molecule, since T4 DNA is also circularly permuted In this way, the process of RDR can in principle initiate soon after an origin-initiated ... proteins to initiate DNA synthesis, and serve as key sites for the regulation of replication timing While the actual determinants of origin activity remain ill defined in many systems, all origins ... recombination proteins in the replication of damaged DNA [1-4] One major set of pathways involves the repair of DSBs and broken replication forks In addition, recombination proteins are involved in multiple...

Ngày tải lên: 11/08/2014, 21:21

... gp59 helicase loading protein, the gp61 primase, and the gp32 single stranded DNA binding protein A.) the gp32 single-stranded DNA binding protein binds to regions of displaced DNA near the replication ... backbone to increase stability of the interaction Cooperative binding of ssb proteins assists in unwinding the DNA duplex at replication forks, recombination intermediates, and origins of replication ... protein, the loading of gp41 helicase requires gp59 helicase loading protein This activity is similar to the E coli DnaC loading of DnaB helicase [70,71] Initially, 59 helicase loading protein...

Ngày tải lên: 11/08/2014, 21:21

... the domains of Vif that are involved in binding to A3G and A3F [12-17] Furthermore, as a proof of principle, work by Mehle et al has shown that Vif peptides overlapping the A3G-binding domain were ... on the ongoing replication of HIV-1 has not been studied in depth Recently, Mulder et al have shown that a replication- competent virus containing mutations in Vif residues involved in interactions ... mutations being the highest in viral DNA, intermediate in cRNA, and lowest in vRNA We also determined the frequency of G-to-A mutations present in vRNA obtained from HIV WT virus infections We...

Ngày tải lên: 13/08/2014, 05:21

... primase DnaG may be involved in DNA replication/ repair in Archaea since the gene encoding DnaG is adjacent to the gene encoding PolB3 in the three crenarchaeal lineages investigated and is located in ... Two main hypotheses can be put forward to explain the genomic association of genes encoding proteins involved in DNA replication and genes coding for proteins involved in translation First, replication ... oligonucleotide/oligosaccharide-binding (OB)-fold containing protein, a recently described new GTPase, DnaG) are encoded by genes that co-localize with DNA replication genes, suggesting that they may be involved in DNA transaction...

Ngày tải lên: 14/08/2014, 08:21

... which recognize and bind to replication origins only during stages of low Cyclin-Cdc28 activity (i.e during G1), and permitting the initiation of replication only during high Cyclin-Cdc28 activity ... mechanism elucidated in the context of DNA damage checkpoint is responsible for preventing untimely spindle extension during vii replication checkpoint-induced S phase arrest We find that although ... Regulation of Spindle Dynamics by DNA Replication Checkpoint 183 6.2 Regulation of Spindle Dynamics by the DNA Damage Checkpoint 186 6.3 A Unified View of DNA Replication and Damage Checkpoints ...

Ngày tải lên: 14/09/2015, 14:03

... residues are involved in the ssDNA binding by bacterial single stranded DNA- binding protein (SSB) and human replication protein A (RPA) [23,24], we performed mutational analyses on Tyr232 in the L1 ... alanine If the aromatic side chain is involved in the interaction with DNA, then its replacement with alanine, a short side chain amino acid residue, should affect the DNA binding of the protein ... residue in the L1 loop is involved in the functional DNA binding during strand exchange Tryptophan-scanning mutagenesis of the HsRad51-L1 loop To gain further information about DNA binding by...

Ngày tải lên: 19/02/2014, 06:20

... and DNA repair mechanisms The functioning of replication proteins, recombination proteins, and checkpoint proteins is closely intertwined During DNA replication, stalled forks generate single-stranded ... chromatin bridges Thus, Geminin may be an example of a metazoanspecific regulator of DNA replication initiation Preventing re -replication When DNA replication is initiated, origin licensing should ... differences in DNA content must be distinguished, e.g when comparing cells in G1 vs S phase In order to investigate if cells go through the process of DNA replication, or whether DNA replication...

Ngày tải lên: 08/03/2014, 19:20

... situated in the interface between the helicase and the protease domains, creating a well-dened binding cleft (Fig 1A) [7] Indeed, it appears that certain residues in the helicase domain may interact ... enzyme, the C-terminus is located in the substrate binding site and the helicase clearly interacts with the C-terminus of NS3 (Fig 1B) [7] By structural modelling of an inhibitor binding to full-length ... domain (red), helicase domain (blue) and NS4A cofactor fused to the N-terminus (green) (PDB: 1CU1) [7] The structural zinc atom in the protease domain and a phosphate group in the helicase domain...

Ngày tải lên: 16/03/2014, 06:20

... decreased in reactions including FEN-1 and dNTPs (Fig 4A) Repair DNA synthesis, displacing the 5¢-dLp residue by Polb alone, did not block the DPC formation, indicating that removal of dL-containing DNA ... 5¢-dRp, which appears to be the late-limiting step in short-patch BER [64], may be critical in determining the mode of BER DNA protein crosslink formation in the short-patch BER of dL Chemical methods ... monofunctional DNA glycosylases [39,41,62,63] As measured by an in vivo assay using a plasmid containing a single AP site in the stop codon of the gene encoding enhanced green fluorescent protein, > 80%...

Ngày tải lên: 16/03/2014, 13:20

... removing the intervening DNA including the lesion The resulting gap is then filled in by DNA polymerase, and the nicks are ligated with DNA ligase (reviewed in [126]) In the case of DNA DSBs, the DNA ... chromatin remodeling in replication may be to help to strategically position nucleosomes close to origins of replication Occlusion of the binding site for the replication initiator, the origin recognition ... modifications include lysine ubiquitination, arginine methylation, and phosphorylation on serine or threonine side chains Histone modifications may affect chromatin structure directly by altering DNA histone...

Ngày tải lên: 16/03/2014, 16:20

... Components in PCR reaction  Template DNA  Primers  dNTPs  Taq DNA polymerase  Buffer  Steps programmed in PCR  Step 1: Denature DNA, 95oC, 5min  Step 2: Annealing: - Denature DNA, 950C, 30s-1min ... Annealing: - Denature DNA, 950C, 30s-1min - Annealing, 55-60oC, 1min  Step 3: Extension, 70-72oC, 30s-1min Go to step 2, 32 cycles  Step 4: Holding, 4oC, 0s  Primers (short DNA fragments) containing ... copies of DNA  Total copies = 2n where n is the number of cycles to copy DNA  So basically it is the cycles of heating and cooling (thermal cycling)  Applications of PCR  DNA cloning  DNA based...

Ngày tải lên: 23/03/2014, 22:20

... protein -DNA structure formed by the initial unwinding of the ORI DNA Owing to its ssDNA binding capability RPA associates with the RC once unwinding of the ORI DNA is underway; here it assists in ... GINS to the Mcm2-7 complex GINS binding increases the helicase activity of the Mcm2-7 hexamer allowing unwinding of duplex DNA The association of GINS also marks a transition when Mcm2-7 binding ... ed into distinct stages The first is the binding and recognition of the origin of replica‐ tion by origin binding proteins For cellular replication in eukaryotes, these proteins are the Origin...

Ngày tải lên: 30/03/2014, 00:20

... (a) studying their cross-linking to newly synthesized DNA, (b) determining their association with chromatin, (c) following the effect of cognate inhibitory antibodies on DNA replication in isolated ... pols exhibited in cross-linking nascent DNA (Fig 1) and binding chromatin (Fig 2), as well as the degree of inhibition of replicative DNA synthesis attained with cognate inactivating antibodies ... Uncoupling from DNA unwinding has been observed in Xenopus extract treated with the polymerase inhibitor aphidicolin [65,66], and uncoupling of leading from lagging strand synthesis after emitine-induced...

Ngày tải lên: 30/03/2014, 10:20

... folding of the minimal DNAbinding domain [50]; substitution of any of its zinccomplexing cysteines (Cys105, Cys108, Cys126 and Cys129) leads to diminished DNA binding and a severe reduction in ... present not just in transcription factors, but in basically all families of proteins involved in maintaining genomic stability, including DNA repair proteins and cell cycle control proteins [18] This ... that is mostly involved in DNA binding but also in protein–protein interactions [17] Originally identified as being present in different transcription factors, it is now known that zinc finger structures...

Ngày tải lên: 30/03/2014, 15:20

... either unmodified or contained a phosphoserine residue in the SQ motif Proteins containing known phosphoserine ⁄ threonine binding motifs such as BRCT or FHA domains may bind to some degree to any ... ‘see’ phosphorylated H2AX in mitotically dividing cells In addition, the binding and accumulation of chromatin-modifying complexes to DNA breaks in yeast was examined in asynchronous haploid cell ... to DNA breaks At least for Ino80, this may involve the Nhp10 protein, identified as important for Ino80-binding chromatin near DNA breaks [30] As the Ino80 complex from an nhp10 mutant strain...

Ngày tải lên: 30/03/2014, 20:20

... properties in vitro It incorporates nucleotides opposite abasic template sites and incorporates chain terminating dideoxy- and arabinonucleotides 11 It contains 3' -exonuclease activity, and its high exonuclease ... No 1822 024) M13Gori DNA, single-stranded binding protein (SSB), and dnaG primase (listed below) are obtained from ENZYCO, Inc., Denver, CO Each 25-tzl reaction contains, in order of addition: ... coli DNA POL II l9 step is given in Table I, and a silver-stained gel showing protein banding patterns and enrichment of Pol II during purification is shown in Fig Purification o f Pol H from Exonuclease- Deficient...

Ngày tải lên: 11/04/2014, 09:40

... digested with DpnI to remove the input DNA The remaining 10% of the linearised DNA was digested with MboI to remove replicated DNA, leaving linearised input DNA (input DNA is resistant to MboI digestion) ... [15] E2 binds to four sites within the HPV LCR and via a protein-protein interaction, increases binding of the viral E1 protein to the origin of replication [16,17] E1 is an ATPdependent helicase ... the HPV 18 E2 protein binds to p53 in cells and the C-terminal domains of the HPV 18 and 16 E2 proteins bind to p53 in vitro, the C-terminal domain of the HPV 11 E2 does not bind to p53 [26] Furthermore,...

Ngày tải lên: 20/06/2014, 01:20