Fig 1.
β-clamp inhibits Pol I-mediated nick translation.
(A) The template used in the assay was prepared by assembling 67 bases, 5’-phosphorylated 28 bases and 5’-radiolabelled (asterisk) 19 bases oligonucleotides. (B) A representative urea denaturing PAGE and the rate calculation therein (see also S2 Fig) indicate that the presence of β-clamp nominally affected nick translation traversing 28 bases downstream RNA. (C) The urea-denaturing gel shows that Pol I efficiently translated the nick, degrading 28 bases downstream DNA, but the presence of β-clamp dramatically reduced the speed of nick translation. The presence of ligase allowed early ligation when β-clamp slowed down Pol I-mediated nick translation. (D) The urea PAGE represents that the 3’-exo- Pol I-mediated nick translation was slowed down in the presence of template-loaded β-clamp, in a manner similar to the nick translation with β-clamp and Pol I combinations, as shown in panel C. Moreover, the presence of template-loaded β-clamp increases the ligation efficiency during the 3’-exo- Pol I-mediated nick translation. (E) The urea denaturing gel represents that the 5’-exo- Pol I intensely paused at the 39th nucleotide and downstream positions, affecting the ligation process. The presence of template-loaded β-clamp moderately enhances these pauses, but facilitates ligation to some extent. The # indicates the position of truncated by-products that originated from the 67-nucleotide long product by the 3’ exonuclease function of Pol I and 5’-exo- Pol I (panel B, C, E). This truncated product is missing in the assay with 3’-exo- Pol I (panel D). All the experiments were performed at least three times. The values represent mean ± standard deviation (sd).
Fig 2.
β-clamp inhibits strand displacement activity of exo- Klenow and exo- Pol I.
(A) The urea PAGE shows that exo- Klenow exhibits a slower speed of strand displacement. Presence of the template-loaded β-clamp stalls the exo- Klenow pauses mostly at the 39th nucleotide position. The exo- Klenow-mediated strand displacement coupled with ligation represents that the DNA ligase fails to ligate nicks. However, the presence of template-loaded β-clamp increases the ligation frequency. (B) exo- Pol I exhibits similar functional consequences on the strand displacement and ligation as observed in case of exo- Klenow (panel A). The values representing mean ± sd are calculated from three different experiments.
Fig 3.
β-clamp inhibits the contact between the 5’end of nicked DNA and Pol-I.
(A) Structural model of E. coli Klenow /DNA complex shows that finger domain of Klenow makes a contact with downstream nick site. The model shows that the conserved F771 (in blue surface representation), which participates in the strand displacement, is positioned between the downstream nicked DNA strands. (B) A fraction of BrdU base containing radiolabelled 28 bases oligonucleotide shows a gel shift under the near UV light, suggesting that the oligonucleotide makes cross-linked adduct with Klenow during strand displacement (lane 2). The presence of β-clamp and γ-complex in the absence of ATP (lane 3), or ATP alone (lane 4) also exhibited similar crosslinking adduct. However, β-clamp, γ-complex, and ATP together suppressed the formation of crosslinking product (lane 6), suggesting that loading of the β-clamp on the template blocks the crosslinking. * Represents a minor contaminated band with the custom synthesized 28 bases oligonucleotide.
Fig 4.
β-clamp increases the flap endonuclease activity of Pol I.
(A) and (B) show the templates assembled using the various lengths of oligonucleotides. Asterisks (*) indicate the radiolabelled 5’-end of the oligonucleotides. (C) A representative denaturing gel indicates that the presence of β-clamp modestly increased the efficiency of single nucleotide cleavage at different time points. Also see S8A Fig for the fraction cleavage rate calculation. (D) A representative denaturing gel shows that β-clamp greatly enhanced the efficiency of 10 nucleotide long flap removal. Also see S8B Fig for fraction cleavage rate calculation. All the experiments were done at least three times. The values represent mean ± sd.
Fig 5.
The model showing influence of β-clamp in gap repair.
(A) The model shows that Pol I removes downstream oligonucleotide by an active nick translation (red flap). The upcoming ligase molecule may ligate the polymerizing upstream (green) and the depolymerising downstream (red) strands occasionally. (B) The clamp bound Pol I has been shown at the nick site. Passive strand displacement for shorter stretch of nucleotides is shown (short red flap). We hypothesize that efficient flap removal of clamp-bound Pol I frequently creates new nick sites. As a result, upcoming ligase molecule may have plenty of time to ligate polymerizing upstream (green) and depolymerising downstream (red) DNA strands at an early stage.