primpol 514–528 peptide  (GenScript corporation)

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) Schematic showing the sequence of PrimPol's RBM-A (residues 510–528), located in the C-terminal RBD (residues 480–560). ( b ) 15 N- 1 H HSQC spectra showing RPA70N in the absence (black) or presence (red) of twofold molar excess of unlabelled RBM-A peptide. ( c ) Electrostatic surface model of RPA70N with RBM-A (green) bound in the basic cleft. Basic and acidic surfaces are coloured blue and red, respectively. ( d ) Key stabilizing interactions of RBM-A (green) in the RPA70N basic cleft (purple). RBM-A binds between β sheets in the β barrel of RPA70N. Of particular importance for binding are the electrostatic interactions of D519 with the side chains of two arginines (R31 and R43) in the RPA70N basic cleft.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Sequencing, Binding Assay

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) The sequence of PrimPol's RBM-B (residues 542–560), located in the C-terminal RBD (residues 480–560). ( b ) The continuous electron density of RBM-B residues 548–556 in the complex with RPA70N. ( c ) Electrostatic surface model of RPA70N with RBM-B (green) bound in the basic cleft. Basic and acidic surfaces are coloured blue and red, respectively. ( d ) 15 N- 1 H HSQC spectra showing RPA70N in the absence (black) or presence (red) of a twofold molar excess of unlabelled RBM-B peptide. ( e ) Key stabilizing interactions of RBM-B (green) in the RPA70N basic cleft (purple). RBM-B binds between β sheets in the β barrel of RPA70N. D551 is of particular importance as it forms a number of electrostatic interactions with both the side chains and a backbone amide NH of the RPA70N peptide.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Sequencing

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) Mutation of RBM-A does not abolish binding of PrimPol's RBD to RPA70N. Chromatographs showing the retention volumes of RBD A-KO (purple), RPA70N (black) and RBD A-KO with RPA70N in a 1:1 ratio (green). ( b ) 15 N- 1 H HSQC spectra showing RPA70N alone (black), in the presence of twofold molar excess of either RBD A-KO (green) or RBM-B peptide (residues 542–560) (red). The perturbations observed for RBD A-KO are similar to those induced by the RBM-B peptide. ( c ) Truncation of RBM-B does not prevent binding of PrimPol's RBD to RPA70N. Chromatographs showing the retention volumes of RBD B-KO (purple), RPA70N (black) and RBD B-KO with RPA70N in a 1:1 ratio (blue). ( d ) 15 N- 1 H HSQC spectra showing RPA70N alone (black) or in the presence of twofold molar excess of RBD B-KO (blue) or RBM-A peptide (residues 510–528) (red). The perturbations observed for RBD B-KO are similar to those induced by the RBM-A peptide. ( e ) Mutation of both RBM-A and RBM-B abolishes the binding of PrimPol's RBD to RPA70N. Chromatographs showing the retention volumes of RBD A/B-KO (purple), RPA70N (black) and RBD A/B-KO with RPA70N in a 1:1 ratio (red). ( f ) 15 N- 1 H HSQC spectra showing RPA70N alone (black) or in the presence of twofold molar excess of RBD A/B-KO (red). The near identity of the two spectra indicates there is no interaction.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Mutagenesis, Binding Assay

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) Schematic detailing the domain architecture of N-terminal FLAG-tagged PrimPol transfected into HEK-293 derivative cells (Flp-In T-Rex-293). The RBD (480–560) containing the RBM-A and B sites is shown below with the mutations forming the A-KO (D519R and F522A) and B-KO (D551R and I554A) highlighted. ( b ) Flp-In T-Rex-293 cells were transfected with wild-type and RBM-A and B mutated PrimPol. Expression was confirmed by addition of 10 ng ml −1 doxycycline (indicated by Dox ± on figure) for 24 h and subsequent western blotting. ( c ) Flp-In T-Rex-293 cells transfected with FLAG-tagged wild-type (WT) PrimPol were grown in the presence or absence of doxycycline (10 ng ml −1 , 24 h), FLAG-PrimPol was immunoprecipitated from the soluble cell lysate using anti-FLAG antibody and western blotted for PrimPol (anti-FLAG) and RPA (anti-RPA2). The presence and absence of doxycycline is indicated by ± Dox, ‘In' indicates the input, ‘E1', ‘E2' and ‘E3', indicate elutions 1, 2 and 3, respectively. ( d ) Immunoprecipitation of FLAG-PrimPol A-KO (D519R/F522A) from Flp-In T-Rex-293 cells grown in the presence and absence of doxycycline. ( e ) Immunoprecipitation of FLAG-PrimPol B-KO (D551R/I554A) from Flp-In T-Rex-293 cells grown in the presence and absence of doxycycline. ( f ) Immunoprecipitation of FLAG-PrimPol A+B-KO (D519R/F522A and D551R/I554A) from Flp-In T-Rex-293 cells grown in the presence and absence of doxycycline.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) PrimPol −/− DT40 cells were complemented with un-tagged human PrimPol constructs; wild-type hPrimPol (+ WT), hPrimPol D519R/F522A (+ A-KO) and hPrimPol D551R/I554A (+ B-KO). ‘WT' indicates lysate from wild-type DT40 cells, ‘−/−‘ indicates lysate from PrimPol −/− DT40 cells. ( b ) DNA fibre analysis was performed on DT40 cells expressing each PrimPol construct. Cells were ultraviolet-C irradiated (20 J m −2 ) between the CldU and IdU labelling periods (each 20 min). Representative DNA fibres showing 1:1, 2:1 and 3:1 CldU:IdU ratios are presented; >100 individual DNA fibres were scored for each experiment. ( c ) Mutation of RBM-A causes increased fork stalling following ultraviolet-C irradiation. Data are representative of the means of three individual experiments and were subject to an unpaired t -test showing a significant difference between the mean CldU/IdU ratio for the ‘+ WT hPrimPol' and ‘+ A-KO hPrimPol' data sets ( P <0.05). ( d ) DNA fibre analysis from the ‘+ A-KO hPrimPol' DT40 cells presented as a cumulative percentage of forks at each ratio. ( e ) Mutation of RBM-B does not significantly alter the level of fork stalling following ultraviolet-C irradiation. DNA fibre analysis of the ‘+ B-KO hPrimPol' DT40 cells, showing the percentage of forks at each CldU:IdU ratio. Data are representative of the means of three individual experiments. ( f ) DNA fibre analysis from the ‘+ B-KO hPrimPol' DT40 cells presented as a cumulative percentage of forks at each ratio.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Construct, Expressing, Irradiation, Mutagenesis

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) PrimPol's RBM-A, but not RBM-B, is critical for recruitment to chromatin. Flp-In T-Rex-293 cells transfected with WT and RBM-A and B mutant PrimPol constructs were either mock (−) or ultraviolet-C (30 J m −2 ) (+) irradiated before separation into Triton X-100 (0.5%) soluble and insoluble fractions. Samples were analysed by western blot alongside whole-cell extracts. Only insoluble samples are presented here, whole-cell extracts and soluble blots can be found in . ( b ) PrimPol's RBD is recruited to Xenopus egg extract chromatin in response to aphidicolin treatment, RBM-A is critical for this recruitment. Recombinant hPrimPol GST constructs (4 ng μl −1 ) were added to Xenopus egg extract supplemented with sperm nuclei (3 × 10 3 μl −1 ). Extract was treated with aphidicolin 100 μg ml −1 and incubated at 21 °C for 80 min. Chromatin was isolated and associated proteins analysed by SDS–PAGE and western blotting using the antibodies indicated. ( c ) Low concentrations of RPA stimulate PrimPol's primase activity, high concentrations inhibit. Primer synthesis by WT hPrimPol (400 nM) on M13 ssDNA templates (20 ng μl −1 ) in the presence of increasing concentrations of RPA. ‘C' indicates the no enzyme control, oligonucleotide (Nt) length markers are shown on the left of the gel. ( d ) Quantification of data shown in ‘c'. For each RPA concentration the fold increase in primer synthesis relative to reactions containing no RPA was calculated. Data are representative of three repeat experiments. ( e ) Schematic showing the effect of increasing RPA concentrations on PrimPol's primase activity. When no RPA is present a proportion of PrimPol binds to the M13 template and facilitates primer synthesis (left). When low/moderate concentrations of RPA are present PrimPol is recruited to the RPA/ssDNA interface causing an increase in primer synthesis activity (middle). At high RPA concentrations the M13 DNA template is fully saturated, blocking access of PrimPol to the DNA and inhibiting primer synthesis (right).

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Transfection, Mutagenesis, Construct, Irradiation, Western Blot, Recombinant, Incubation, Isolation, SDS Page, Activity Assay, Control, Concentration Assay, Blocking Assay

Journal: Nature Communications

Article Title: Molecular basis for PrimPol recruitment to replication forks by RPA

doi: 10.1038/ncomms15222

Figure Lengend Snippet: ( a ) Unrepaired DNA damage lesions, or DNA secondary structures, in the leading strand template lead to stalling of polymerase ɛ. This causes uncoupling of replication, generating ssDNA downstream of the DNA damage lesion/structure and facilitating binding of RPA. Note that for simplicity other replisome components and lagging strand synthesis machinery are not shown. ( b ) PrimPol is recruited to the ssDNA interface uncovered by the replicative helicase through the interaction of its RBM-A with RPA70N. This interaction is stabilized by the binding of the ZnF and AEP domains to ssDNA. ( c ) PrimPol catalyses the synthesis of a new DNA primer, before further extension is prevented by the restraining effect of the RPA interaction and ZnF domain, coupled with the enzyme's low processivity. ( d ) Unable to continue with primer extension, PrimPol dissociates from the template strand. Re-binding upstream is prevented by RPA. ( e ) The nascent primer is utilized by the replicative polymerase for continued DNA replication. This leaves behind a short RPA-coated ssDNA region opposite the lesion to be filled in by template switching or TLS.

Article Snippet: The PrimPol 514–528 peptide used for co-crystallization experiments was synthesized (Genscript) and used as supplied

Techniques: Binding Assay