α pol κ  (Bethyl)

Journal: Science Advances

Article Title: Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA syntheses

doi: 10.1126/sciadv.adw4618

Figure Lengend Snippet: ( A ) Quantitative real-time PCR of PrimPol and Pol κ normalized to GAPDH in U2OS cells. Error bars represent the SD of three technical replicates from one experiment representative of two independent experiments. P < 0.01. ( B ) IdU track lengths from DNA fibers in U2OS cells. Cells were labeled with CldU and IdU for 10 and 30 min, respectively. Approximately 300 DNA fibers obtained from three independent experiments were analyzed for each condition. The bars on top of the distribution clouds indicate the median. P < 0.001. ( C ) Western blot revealing PrimPol and Pol κ levels in U2OS cells with the indicated siRNAs. Actin was used as a loading control. ( D ) Representative image of U2OS cells transfected with the V5-PrimPol expression plasmid with (positive) or without (negative) V5-PrimPol foci (scale bar, 5 μm). ( E and F ) Percentage (means + SD) of U2OS cells with V5-PrimPol foci. Approximately 750 nuclei per sample were analyzed in three independent experiments. P < 0.01. ( G ) Percentage (means + SD) of U2OS cells with V5- PrimPol or V5-PrimPol RBDm foci. Approximately 600 nuclei per sample were analyzed in two independent experiments. P < 0.01. ( H ) Percentage (means + SD) of U2OS cells with more than four RPA foci. Approximately 300 nuclei per sample were analyzed in two independent experiments. P < 0.01. The right panel shows representative images of U2OS cells with less than four RPA foci (negative) or with more than four RPA foci (positive). Scale bar, 10 μm. ( I ) Model. Partial silencing of p21 causes an increase in PrimPol-dependent nascent DNA synthesis. In contrast, complete silencing of p21 causes an augmentation of Pol κ–mediated DNA replication.

Article Snippet: The following antibodies were used: α-p21 1:1000 (Santa Cruz Biotechnology, sc-6246, RRID: AB_628073), α-p53 1:1:1 (phosphate-buffered saline:purified immunoglobulin from clone DO-1:purified immunoglobulin from clone PAB-1801), α-Pol η 1:1000 (Santa Cruz Biotechnology, sc-5592, RRID: AB_2167006), α-Pol κ 1:1000 (Bethyl Laboratories, A301-977A, RRID: AB_1548020), α-PrimPol 1:1000 (Proteintech 29824-1-AP, RRID: AB_2918349), and α-Actin 1:20000 (Sigma-Aldrich, A2066, RRID: AB_476693).

Techniques: Real-time Polymerase Chain Reaction, Labeling, Western Blot, Control, Transfection, Expressing, Plasmid Preparation, DNA Synthesis

Journal: Science Advances

Article Title: Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA syntheses

doi: 10.1126/sciadv.adw4618

Figure Lengend Snippet: ( A ) Western blot revealing p53 and p21 in parental HCT116, HCT116 p53 KO, and HCT116 p21 KO cells. Actin was used as a loading control. ( B ) Quantitative real-time PCR of PrimPol and Pol κ normalized to GAPDH. Error bars represent the SD of three technical replicates within one experiment from two independent experiments. P < 0.01. ( C ) IdU track lengths from DNA fibers in HCT116, HCT116 p53 KO, and HCT116 p21 KO cells. CldU: 10 min; IdU: 30 min. Approximately 300 DNA fibers obtained from three independent experiments were analyzed for each condition. The bars indicate the median. P < 0.001. ( D ) IdU track lengths from DNA fibers in HCT116, HCT116 p53 KO, and HCT116 p21 KO cells. CldU: 10 min; IdU: 30 min. Approximately 200 DNA fibers obtained from two independent experiments were analyzed for each condition. The bars indicate the median. P < 0.001. The lower panel shows a Western blot revealed with a p21 antibody. Actin was used as a loading control. ( E ) IdU track lengths from DNA fibers in U2OS cells. CldU: 10 min; IdU: 30 min. Approximately 150 DNA fibers obtained from two independent experiments were measured for each condition. The bars indicate the median. P < 0.001. The lower panel shows p53 and p21 levels in a Western blot. Actin was used as a loading control. ( F ) IdU track lengths from DNA fibers in U2OS WT (clone 18) and U2OS Pol ι KO (clones 13 and 24) cells. CldU: 10 min; IdU: 30 min. Approximately 200 DNA fibers obtained from two independent experiments were analyzed for each condition. The bars indicate the median. P < 0.001. ( G ) Model. p21 regulates PrimPol through Pol ι. However, p21 regulates Pol κ more strongly, and it can only synthesize DNA when p21 is completely depleted.

Article Snippet: The following antibodies were used: α-p21 1:1000 (Santa Cruz Biotechnology, sc-6246, RRID: AB_628073), α-p53 1:1:1 (phosphate-buffered saline:purified immunoglobulin from clone DO-1:purified immunoglobulin from clone PAB-1801), α-Pol η 1:1000 (Santa Cruz Biotechnology, sc-5592, RRID: AB_2167006), α-Pol κ 1:1000 (Bethyl Laboratories, A301-977A, RRID: AB_1548020), α-PrimPol 1:1000 (Proteintech 29824-1-AP, RRID: AB_2918349), and α-Actin 1:20000 (Sigma-Aldrich, A2066, RRID: AB_476693).

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction, Clone Assay

Journal: Science Advances

Article Title: Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA syntheses

doi: 10.1126/sciadv.adw4618

Figure Lengend Snippet: ( A ) Percentage (means + SD) of U2OS cells with V5-PrimPol foci. Approximately 500 nuclei per sample were analyzed in two independent experiments. P < 0.01. ( B ) Western blot revealing Pol η and p21 in parental HCT116 and HCT116 p21 KO cells. Actin was used as a loading control. ( C ) Percentage (means + SD) of HCT116 and HCT116 p21 KO cells with V5-PrimPol foci. Approximately 500 nuclei per sample were analyzed in two independent experiments. P < 0.01. ( D ) IdU track lengths from DNA fibers in HCT116 and HCT116 p21 KO cells. Cells were labeled with CldU and IdU for 10 and 30 min, respectively. Approximately 200 DNA fibers obtained from two independent experiments were analyzed for each condition. The bars on top of the distribution clouds indicate the median. P < 0.001. ( E ) Model. p21 controls PrimPol through Pol ι. However, at high levels, p21 primarily antagonizes Pol κ. Only when p21 is completely depleted, Pols κ and η prevent PrimPol DNA replication.

Article Snippet: The following antibodies were used: α-p21 1:1000 (Santa Cruz Biotechnology, sc-6246, RRID: AB_628073), α-p53 1:1:1 (phosphate-buffered saline:purified immunoglobulin from clone DO-1:purified immunoglobulin from clone PAB-1801), α-Pol η 1:1000 (Santa Cruz Biotechnology, sc-5592, RRID: AB_2167006), α-Pol κ 1:1000 (Bethyl Laboratories, A301-977A, RRID: AB_1548020), α-PrimPol 1:1000 (Proteintech 29824-1-AP, RRID: AB_2918349), and α-Actin 1:20000 (Sigma-Aldrich, A2066, RRID: AB_476693).

Techniques: Western Blot, Control, Labeling

Journal: Science Advances

Article Title: Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA syntheses

doi: 10.1126/sciadv.adw4618

Figure Lengend Snippet: ( A ) Western blot revealing p53 and p21 levels in H1299 and SKOV3 cells. Actin was used as a loading control. sip21#1 (100 nM) was transfected into each cell line to achieve complete p21 down-regulation. ( B ) Quantitative real-time PCR of PrimPol and Pol κ normalized to GAPDH in H1299 and SKOV3 cells. Error bars represent the SD of three technical replicates from one experiment representative of two independent experiments. P < 0.01. ( C ) IdU track lengths from DNA fibers in H1299 cells. Cells were labeled with CldU and IdU for 10 and 30 min, respectively. Approximately 200 DNA fibers obtained from two independent experiments were analyzed for each condition. The bars on top of the distribution clouds indicate the median. P < 0.001. ( D ) Percentage (means + SD) of binucleated H1299 cells with micronuclei. Approximately 300 binucleated cells per sample were analyzed in two independent experiments. P < 0.01. ( E ) Percentage (means + SD) of binucleated H1299 cells with micronuclei. sip21#1 (100 nM) was transfected to achieve complete p21 down-regulation. Approximately 300 binucleated cells per sample were analyzed in two independent experiments. P < 0.01. ( F to H ) The IdU track length from DNA fibers and the percentage of binucleated SKOV3 cells with micronuclei treated with siRNAs were determined as in (C) to (E). P values for (F) to (H) are <0.001, 0.01, and 0.01, respectively.

Article Snippet: The following antibodies were used: α-p21 1:1000 (Santa Cruz Biotechnology, sc-6246, RRID: AB_628073), α-p53 1:1:1 (phosphate-buffered saline:purified immunoglobulin from clone DO-1:purified immunoglobulin from clone PAB-1801), α-Pol η 1:1000 (Santa Cruz Biotechnology, sc-5592, RRID: AB_2167006), α-Pol κ 1:1000 (Bethyl Laboratories, A301-977A, RRID: AB_1548020), α-PrimPol 1:1000 (Proteintech 29824-1-AP, RRID: AB_2918349), and α-Actin 1:20000 (Sigma-Aldrich, A2066, RRID: AB_476693).

Techniques: Western Blot, Control, Transfection, Real-time Polymerase Chain Reaction, Labeling

Journal: Science Advances

Article Title: Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA syntheses

doi: 10.1126/sciadv.adw4618

Figure Lengend Snippet: ( A ) Left panel: The endogenous levels of p21 in cells undergoing DNA replication inhibit Pol κ and PrimPol participation in nascent DNA elongation, ensuring a safe rate of DNA replication (green region in the speed meter). Genomic stability is at its highest. Middle panel: Partial depletion of p21 increases the nascent DNA elongation rate because of excess PrimPol participation in DNA replication (dangerously high region in the speed meter). PrimPol-mediated DNA replication does not induce replication stress but induces genomic instability. Right panel: Complete elimination of p21 reduces the nascent DNA elongation rate because of excess participation of Pol κ in DNA replication. Pol κ–mediated DNA replication triggers replication stress and genomic instability. ( B ) The participation of PrimPol in DNA synthesis is subject to strong regulation. p21 prevents PrimPol from participating in nascent DNA synthesis in concert with the Pol ι/p53 DNA replication complex. When p21 is completely down-regulated, Pols κ and η act in concert to prevent PrimPol-dependent DNA synthesis. Only when p21 is partially down-regulated, a window of opportunity for PrimPol is created because the Pol ι/p53 complex is not favored and the participation of Pols κ and η in DNA replication is still prevented by p21.

Article Snippet: The following antibodies were used: α-p21 1:1000 (Santa Cruz Biotechnology, sc-6246, RRID: AB_628073), α-p53 1:1:1 (phosphate-buffered saline:purified immunoglobulin from clone DO-1:purified immunoglobulin from clone PAB-1801), α-Pol η 1:1000 (Santa Cruz Biotechnology, sc-5592, RRID: AB_2167006), α-Pol κ 1:1000 (Bethyl Laboratories, A301-977A, RRID: AB_1548020), α-PrimPol 1:1000 (Proteintech 29824-1-AP, RRID: AB_2918349), and α-Actin 1:20000 (Sigma-Aldrich, A2066, RRID: AB_476693).

Techniques: DNA Synthesis

Journal: The Journal of Biological Chemistry

Article Title: Dynamic stem–loop extension by Pol θ and templated insertion during DNA repair

doi: 10.1016/j.jbc.2024.107461

Figure Lengend Snippet: Pol θ extends ssDNA and ssRNA oligonucleotides to give distinct products. A , protein domains and constructs of human Pol θ. B , extension of ssDNA oligonucleotide DCM1 using Kf exo - , Pol θ QM1, Pol θ ΔCEN, and Pol θ full-length protein. C , extension of DCM2 ssRNA by Pol θ QM1 ( top ), Pol θ ΔCEN ( middle ), but not Kf exo - ( bottom ). D , densitometric quantification of the bands in Figures 1 C and D plotted together as percent extension versus time ± SD. E , primer/template extension of DNA/DNA ( left ) DCM5/DCM3 and DNA/RNA ( right ) DCM5/DCM3 RNA substrates by Pol θ ΔCEN ( top ) and Kf exo - ( bottom ). F , quantification of the bands in Figures 1 E and C for each enzyme plotted together as percent extension versus time ± SD. Two separate primer/template substrates were used to test primer extension and plotted together. Conditions for all reactions are as follows: 25 nM DNA or RNA, 125 nM enzyme, and 100 μM dNTPs. dNTP, deoxynucleotide; Kf exo - , Klenow fragment exo - .

Article Snippet: Human DNA Pol κ (Enzymax Cat. No. 27).

Techniques: Construct

Journal: The Journal of Biological Chemistry

Article Title: Dynamic stem–loop extension by Pol θ and templated insertion during DNA repair

doi: 10.1016/j.jbc.2024.107461

Figure Lengend Snippet: Extension of short ssDNA oligonucleotides occurs largely by stem–loop synthesis. A , two oligonucleotides were analyzed, in reaction mixtures with 125 nM Pol θ QM1, 25 nM DNA, and 100 μM dNTP. DCM6 is 5′-labeled with Cy5. DCM6-P is 5ʹ-FAM-labeled and has a 3ʹ-phosphate group to prevent extension. If DCM6 extends by using another oligonucleotide as template, ∼two-thirds of the products would migrate on a native gel as a 14- to 17-mer double-stranded species containing both fluorophores. B , denaturing 20% PAGE gel showing that DCM6 produces an extension product migrating as expected and that DCM6-P does not extend. C , nondenaturing 25% PAGE gel showing that extension of DCM6 ( green ) does not arise by pairing with DCM6-P ( purple ), as a paired product would migrate at the 14-bp marker position and contain both fluorophores ( black ). This marker is the annealed product of DCM6 with its complementary FAM-labeled DCM22. D , scheme of predicted products derived from DCM17 and DCM17-4 oligonucleotides. Unimolecular and bimolecular products with potential Pvu I cleavage sites are shown. If bimolecular pairing were used between DCM17 and DCM17-4, DNA synthesis would yield a 41-bp double strand fragment. Pvu I cleavage would yield a ∼30-mer Cy5-labeled fragment and a ∼13-mer FAM-labeled fragment. The colored text shows the original oligonucleotide sequence with the predicted synthesis in gray text . E , DCM17, DCM17-4, or both combined were incubated in reaction mixtures containing 125 nM Pol θ QM1, 25 nM each DNA, and 100 μM dNTPs. On a denaturing 20% polyacrylamide gel, 41-nt products are produced as expected, with the DCM17-4 product susceptible to Pvu I cleavage. F , on a nondenaturing 25% PAGE gel, the samples migrate similarly. There is no 41-bp species, and no Cy5-labeled 30-mer is produced following Pvu I cleavage. This shows that the visible products are synthesized in independent unimolecular reactions. dNTP, deoxynucleotide.

Article Snippet: Human DNA Pol κ (Enzymax Cat. No. 27).

Techniques: Labeling, Marker, Derivative Assay, DNA Synthesis, Sequencing, Incubation, Produced, Synthesized

Journal: The Journal of Biological Chemistry

Article Title: Dynamic stem–loop extension by Pol θ and templated insertion during DNA repair

doi: 10.1016/j.jbc.2024.107461

Figure Lengend Snippet: Factors governing stem–loop extension. A , stem–loop extension is suppressed by altering preferred base-pairing sites ( top ). Quantification of the percent of stem–loop extension for each substrate at different time points ( bottom ). Conditions: 25 nM DNA, 125 nM Pol θ QM1, and 100 μM dNTPs. B , Pol θ QM1 stem – loop extension of DCM9 using different combinations of dNTPs in a 1-min reaction and a longer 10-min reaction. Schematic of hairpins used by Pol θ QM1 in the short and longer reactions. C , Pol θ QM1 stem – loop extension of DCM10 using different combinations of dNTPs in a 10-min reaction ( left ). Conditions: 25 nM DNA, 125 nM Pol θ QM1 (except lane 1, no enzyme), and 100 μM each of the indicated dNTPs. Schematic of the predicted hairpins used by Pol θ QM1 to extend in the reactions containing just dGTP or dCTP ( right ). D , stem–loop extension of DCM1 ssDNA ( top ) and a primer template substrate ( bottom ) using Pol θ QM1 and Pol θ QM1 3A variant. Conditions: 25 nM DNA, 50 nM Pol θ QM1 or 3A variant, and 100 μM dNTPs. E , densitometry quantification of the gel bands in ( D ) for each enzyme plotted as percent stem–loop extension versus time in seconds for the reactions using ssDNA ( top ) and the primer-template ( bottom ). The experiment was run three times, and the combined data were plotted as described in Experimental procedures. With primer-template substrate K half was the same for the two enzymes, but with stem–loop substrate K half = 38 for Pol θ QM1 and 209 for Pol θ QM1 3A, a time lag of 171 s (∼3 min). dNTP, deoxynucleotide.

Article Snippet: Human DNA Pol κ (Enzymax Cat. No. 27).

Techniques: Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Dynamic stem–loop extension by Pol θ and templated insertion during DNA repair

doi: 10.1016/j.jbc.2024.107461

Figure Lengend Snippet: Stem–loop extension activity on ssDNA is confined to specific specialized DNA polymerases . A , stem–loop extension of DCM1 ( left ) and primer-template ( right ) using several DNA polymerases, Pol θ QM1, Pol θ ΔCEN, Pol ν, Pol γ, RB69 (exo - ), Pol η, Pol κ, Pol ι, and Pol β. Conditions: 25 nM DNA, 125 nM enzyme, and 100 μM dNTPs. Reactions were incubated for 10 min at 37 °C. B , stem–loop extension of DCM1 ( left ) and primer-template ( right ) using Pol θ ΔCEN, Pol δ, and Pol λ at various time points. C , stem–loop extension of DCM1 using Pol θ QM1, Pol η, and Pol λ in the presence of different combinations of dNTPs in a long 10-min reaction. Conditions: 25 nM DNA, 125 nM Pol δ/Pol θ QM1/Pol λ, and 100 μM dNTPs. D , stem–loop extension slippage activity by Pol θ ΔCEN ( left ) and Pol η ( right ). Conditions: 25 nM DNA, 125 nM enzyme, and 100 μM dGTP. dNTP, deoxynucleotide; RPA, replication protein A; TMEJ, theta-mediated end joining.

Article Snippet: Human DNA Pol κ (Enzymax Cat. No. 27).

Techniques: Activity Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Dynamic stem–loop extension by Pol θ and templated insertion during DNA repair

doi: 10.1016/j.jbc.2024.107461

Figure Lengend Snippet: Competition between TMEJ and stem–loop extension. A , TMEJ activity assay using Cy5-labeled DCM12 and Cy3-labeled DCM13. The two 90-mers have a 6-nt 3′ microhomology. DNA was either preincubated (lanes P) with each enzyme for 10 min at 37 °C prior to addition of dNTPs or not preincubated (lanes N). Stem–loop extension using Pol θ QM1 (lanes 2 and 5) or Pol θ ΔCEN (lanes 3 and 6) with each individual 90-mer. TMEJ activity using Pol θ QM1 lanes (lanes 7–9) and Pol θ ΔCEN (lanes 10–12). Conditions: 50 nM of each oligo, 100 nM Pol θ QM1 or Pol θ ΔCEN, and 50 μM dNTPs. Gel was scanned for each fluorescent emission and the images were then merged and artificially colored using ImageJ software ( https://imagej.net/software/imagej/ ). Cyan reflects Cy5 gel scan, purple reflects the Cy3 gel scan, and dark blue is the result of superimposed oligonucleotides and acts a read out for TMEJ. Densitometry quantification of fraction of products formed by Pol θ QM1 and Pol θ ΔCEN is labeled at the bottom . B , TMEJ activity of DCM14/DCM14Temp and DCM15/DCM15Temp using Pol θ ΔCEN. Conditions: 50 nM of each oligo, 100 nM Pol θ ΔCEN, and 50 μM dNTPs. DCM14 and DCM15 are 36-mer oligonucleotides with a 6-nt microhomology at the 3′-end. DCM14 has 12 consecutive C nucleotides and DCM15 has 12 consecutive T nucleotides prior to the 6-nt microhomology, which limits stable stem–loop extension pairing sites. Colors as in panel A except FAM has the same color as Cy3. Densitometry quantification of fraction of products formed Pol θ ΔCEN is labeled at the bottom . C , schematic of Cy5 5′-labeled 27-mer (DCM16-2) for TMEJ versus stem–loop extension competition assay. Potential products formed by Pol θ are labeled as a TMEJ product (EJ) in dark blue , a stem–loop extension product (SL) in purple , and a terminated TMEJ product (tEJ) in red . D , denaturing gel showing products of TMEJ and stem–loop extension in a mixture of T-27-mer (DCM16-2) and G-27-mer (DCM17). The dotted line following lane 2 indicates where four lanes were spliced from the gel. Conditions: 25 nM of each oligonucleotide, 200 nM Pol θ ΔCEN, and 100 μM dNTPs/ddNTPs. Densitometry quantification of the fraction of products formed by Pol θ ΔCEN is labeled at the bottom . E , TMEJ versus SL competition following preincubation for 15 min at 37 °C with Pol θ ΔCEN. Preincubation of DCM16-2 with Pol θ ΔCEN, before addition of dNTPs ( left three lanes ) enhanced TMEJ activity. Preincubation of DNA with dNTPs before addition of Pol θ ΔCEN enhanced stem–loop extension at the expense of TMEJ. dNTP, deoxynucleotide; TMEJ, theta-mediated end joining.

Article Snippet: Human DNA Pol κ (Enzymax Cat. No. 27).

Techniques: Activity Assay, Labeling, Software, Competitive Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Dynamic stem–loop extension by Pol θ and templated insertion during DNA repair

doi: 10.1016/j.jbc.2024.107461

Figure Lengend Snippet: RPA inhibits stem–loop extension and TMEJ. A , stem–loop extension of 50-mer ssDNA (DCM18, 6.4 nM) using Pol θ ΔCEN and Pol θ QM1 in the presence of increasing RPA (8, 16, 32, 64, and 128 nM). Conditions: 6.4 nM DNA, 64 nM Pol θ QM1, or Pol θ ΔCEN and 4 μM dNTPs. DNA and RPA were preincubated for 15 min at 37 °C, before addition of Pol θ ΔCEN ± 1 mM ATP and dNTPs (4 μM). B , TMEJ activity assay of two 90-mer paired oligonucleotides (DCM12 and DCM13, 25 nM each) coated with RPA. DNA (25 nM) and increasing concentrations of RPA (0, 0.1, 0.2, 0.4, and 0.8 μM) were preincubated for 15 min at 37 °C, before addition of 200 nM Pol θ ΔCEN ± 5 mM ATP. Reactions were prepared with 100 μM dNTPs ( left ) or 100 μM dTTP/dGTP/dCTP ( right ). dNTP, deoxynucleotide; RPA, replication protein A; TMEJ, theta-mediated end joining.

Article Snippet: Human DNA Pol κ (Enzymax Cat. No. 27).

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Dynamic stem–loop extension by Pol θ and templated insertion during DNA repair

doi: 10.1016/j.jbc.2024.107461

Figure Lengend Snippet: Reconstitution of a TMEJ product incorporating an inverted repeat. Schematic ( A ) and gel scan ( B ) of Cy5-labeled DCM10 and FAM-labeled DCM19 oligonucleotides. The two oligonucleotides do not contain microhomologies sufficient for TMEJ (lanes 2 and 6). DCM10 was extended with Pol θ QM1 for 3 min in the presence of dGTP. Pol θ ΔCEN can use the newly extended DCM10 to pair with DCM19 to effectuate TMEJ (lanes 4 and 8). Conditions: stem–loop extension: 8 nM DCM10, 100 nM Pol θ QM1, and 50 μM dGTP. TMEJ: 8 nM SL-extended DCM10, 8 nM DCM19, 400 nM Pol θ ΔCEN, and 100 μM dNTPs. C , model describing how Pol θ can use stem–loop extension to create a microhomology to allow TMEJ in some situations. RPA binding to ssDNA to block stem–loop extension and TMEJ. The HLD ( cyan ) of Pol θ ΔCEN can use ATP or dATP to remodel the placement of RPA, restoring TMEJ and limited stem–loop extension activity. When the 3′-end cannot be used for end joining, the polymerase ( pink ) folds the 3′-end inside the active site and finds a preferred two-terminal base pairing upstream of the same strand to initiate stem–loop extension and synthesize a short sequence of DNA. Pol θ then releases the hairpin and uses the newly created 3′-end to search the other oligo for a MH site it can use as a primer to perform TMEJ. Repair products will then contain an inverted repeat, representing the newly created 3′-end. dNTP, deoxynucleotide; RPA, replication protein A; TMEJ, theta-mediated end joining.

Article Snippet: Human DNA Pol κ (Enzymax Cat. No. 27).

Techniques: Labeling, Binding Assay, Blocking Assay, Activity Assay, Sequencing