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Results: 6

1.
Fig. 5

Fig. 5. Levels of pol IV corresponding to RpoS-induced cells allow it to limit pol II exonuclease activity at D-loops. From: Preferential D-loop Extension by a Translesion DNA Polymerase Underlies Error-Prone Recombination.

RDR was performed for 30 min with 437.5 nM pol II (lane 1), 6.25 µM of pol IV (lane 2), and pol II and pol IV together (lane 3). Schematic representation of results illustrates limited activity of pol II at D-loops (right). β-clamp, clamp-loader, and SSB were present in all reactions.

Richard T. Pomerantz, et al. Nat Struct Mol Biol. ;20(6):748-755.
2.
Fig. 6

Fig. 6. Model of translesion DNA polymerase activity at D-loops during stress. From: Preferential D-loop Extension by a Translesion DNA Polymerase Underlies Error-Prone Recombination.

Upregulation of pol IV by the SOS and RpoS stress responses enables it to outcompete other pols and play a dominant role in RDR during stress which facilitates error-prone recombination. Pol II, however, intermittently competes with pol IV through its exonuclease domain. D-loop dependent stimulation of pol II exonuclease activity enables the polymerase to move in reverse and partially resect the extended D-loop. This activity likely contributes to proofreading of pol IV errors and suppresses error-prone recombination. Pol IV finally regains access to the D-loop by displacing pol II from the DNA.

Richard T. Pomerantz, et al. Nat Struct Mol Biol. ;20(6):748-755.
3.
Fig. 2

Fig. 2. High levels of pol IV comparable to SOS-induced cells facilitate its recruitment to D-loops. From: Preferential D-loop Extension by a Translesion DNA Polymerase Underlies Error-Prone Recombination.

(a) Schematic of PriA inhibition of D-loop extension by pol III (left). RDR was performed with pol III and the indicated concentrations of PriA (right). (b) RDR was performed with pol IV concentrations corresponding to non-SOS (left panel) and SOS (right panel) conditions in the presence (lanes 2) and absence (lanes 1) of relative cellular levels of PriA (175 nM). Models of competition between pol IV and PriA at D-loops during non-stressed and stressed conditions (right). Relative D-loop extension (RE) was determined as in Fig. 1. β-clamp, clamp-loader, and SSB were present in all reactions.

Richard T. Pomerantz, et al. Nat Struct Mol Biol. ;20(6):748-755.
4.
Fig. 4

Fig. 4. Pol II requires a functional exonuclease domain to compete with pol IV at D-loops. From: Preferential D-loop Extension by a Translesion DNA Polymerase Underlies Error-Prone Recombination.

(a) A timecourse of RDR was performed with pol II (lanes 1–3), pol IV (lanes 4–6), and pol II and pol IV together (lanes 7–9) at relative concentrations corresponding to SOS-induced cells with 50 µM (left panel) or 10 µM (right panel) dNTPs. Schematic representation of results illustrates competition between pol II and pol IV at D-loops (right). (b) A timecourse of RDR was performed with exonuclease deficient pol II (lanes 1–3), pol IV (lanes 4–6), and exonuclease deficient pol II and pol IV together (lanes 7–9) at relative concentrations corresponding to SOS-induced cells with 50 µM (left) or 10 µM (right) dNTPs. Schematic representation of results illustrates the inability of exonuclease deficient pol II to compete with pol IV at D-loops (right). (c) RDR was performed with exonuclease deficient pol II (lane 1), pol IV (lane 2), and exonuclease deficient pol II and pol IV together (lane 3) at relative concentrations corresponding to SOS-induced cells with 50 µM dNTPs. β-clamp, clamp-loader, and SSB were present in all reactions.

Richard T. Pomerantz, et al. Nat Struct Mol Biol. ;20(6):748-755.
5.
Fig. 3

Fig. 3. Pol II switches to an active exonuclease mode at D-loops. From: Preferential D-loop Extension by a Translesion DNA Polymerase Underlies Error-Prone Recombination.

(a) A timecourse of RDR was performed with wild-type (left) and exonuclease deficient (right) pol II. (b) A timecourse of replication was performed by wild-type and exonuclease deficient pol II on a linear double-strand DNA template (left) and a circular primer-template (right). (c) RDR was performed with wild-type pol II in the presence (lane 2) and absence (lane 1) of gyrase. (d) Model of pol II activity at D-loops. 1. Pol II engages its polymerase mode to extend a D-loop. 2. Pol II pauses due to superhelical tension in the DNA. 3. Superhelical tension in the DNA promotes reverse translocation and exonuclease activity of pol II. 4. Pol II switches to a highly active exonuclease mode. (e) A timecourse of RDR was performed with wild-type pol II and 10 µM dNTPs. (f) RDR was performed with pol II and 50 µM dNTPs for 15 min, the reaction was then divided and aliquots were incubated for a further 5 min in the presence (lane 2) or absence (lane 1) of 100 µM dNTPs. (g) A timecourse of RDR was performed with wild-type pol II and 100 µM dNTPs. (h) A timecourse of RDR was performed with wild-type (lanes 1–3) or exonuclease deficient (lanes 4–6) pol III and 50 µM dNTPs. (i) A timecourse of replication by wild-type (black) and exonuclease deficient (grey) pol III holoenzyme (pol III, β) was performed with 50 µM dNTPs on a m13 primer-template substrate. DNA products were analyzed in a denaturing alkaline agarose gel and analyzed by phosphorimager. (j) The replisome containing DnaB, β, τ-complex and either wild-type (red circles) or exonuclease deficient (grey circles) pol III was assembled on a rolling circle template immobilized to streptavidin beads in the presence of dGTP and dCTP. Unbound proteins except for β were removed by washing then a timecourse of leading strand synthesis was initiated by adding dATP, 32P-α-dTTP and SSB. DNA products were analyzed as in (i). (k) Pol III (lanes 2–6) or pol II (lanes 8–12) was incubated with a radio-labeled primer-template with (right) or without (left) a mismatch in the absence of dNTPs for the indicated times. DNA products were resolved in a denaturing gel. β-clamp, clamp-loader, and SSB were present in all reactions.

Richard T. Pomerantz, et al. Nat Struct Mol Biol. ;20(6):748-755.
6.
Fig. 1

Fig. 1. Pol IV is highly proficient and error-prone in recombination-directed replication. From: Preferential D-loop Extension by a Translesion DNA Polymerase Underlies Error-Prone Recombination.

(a) Model of DSB repair. DNA ends are resected by nucleases resulting in 3’ ssDNA tails. RecA promotes strand invasion resulting in a D-loop. Pol extends the D-loop (red arrow). The second DNA end is captured then Holliday junctions are formed which are subsequently resolved by an endonuclease. (b) Scheme for reconstitution of RDR (D-loop extension). A 5’-32P labeled ssDNA is incubated with RecA, ATP and dNTPs which promotes RecA filament formation. A supercoiled plasmid containing the same sequence as the ssDNA is then added which facilitates D-loop formation. The β-clamp, which confers processivity onto pols, is then assembled at the D-loop by adding β along with its clamp-loader (γ-complex) and SSB. Last, DNA polymerase is added which initiates RDR by extending the D-loop. (c) RDR was performed with 500 nM pol IV (lanes 1–4) or pol V (lanes 5–8) for the indicated times. (d) Controls for pol IV RDR activity. RDR was performed as in (c) in the presence or absence of the indicated reagents. (e) RDR was performed with 500 nM pol V in the presence of increasing amounts of ssDNA and 3.3 µM RecA. (f) Primer extension was performed with 500 nM pol V and 2 µM RecA in the presence (lane 3) and absence (lane 2) of 160 nM trans ssDNA. * indicates 32P. (g) RDR was performed with pol IV at relative concentrations corresponding to SOS-induced cells in the presence (lane 3, left) and absence (lane 2, left; right) of β with (right) or without (left) increasing amounts of sodium glutamate (NaGlu). Relative D-loop extension (RE) was determined by dividing the fraction of D-loop extension observed in lane 2 by that observed in lane 3 (left). Fraction of D-loop extension was determined by dividing the intensity of the extended D-loop product by the sum of the intensities of the unextended and extended D-loop products. (h) Primer (left) and D-loop (right) extension were performed with pol IV and the indicated dNTP. D-loops were purified and DNA products were resolved in denaturing urea polyacrylamide gels. RE was determined by dividing the fraction of extension products for each lane by the fraction of extension products in lane 2 for each panel. (i) D-loop extension was performed as in (h). (j) RDR was performed with pol IV in the presence of 50 µM dGTP and 10 µM 2',3'-dideoxyadenosine triphosphate (ddATP)(lane 1). Incorporation of the ddAMP chain terminator opposite the thymidine base (T) prevents further extension of the D-loop. DNA products were analyzed as in (h). The DNA sequence of the product in lane 1 was determined by comparison to the DNA markers in lanes 2 and 3. The mobility of the product in lane 1 (upper band) corresponds to the marker in lane 2 which indicates that the D-loop was extended by the incorporation of 3 dGMPs and 1 ddAMP demonstrating a −1 frameshift mutation (see schematic at bottom). Partial DNA sequences of the invading ssDNA and markers are indicated. β-clamp, clamp-loader, and SSB were present in all reactions except where indicated.

Richard T. Pomerantz, et al. Nat Struct Mol Biol. ;20(6):748-755.

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