Escherichia Coli Β-Clamp Slows Down DNA Polymerase I Dependent Nick

bioRxiv preprint doi: https://doi.org/10.1101/256537; this version posted June 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

2 Escherichia coli -clamp slows down DNA polymerase I dependent nick translation while

3 accelerating ligation

5 Amit Bhardwaj1, Debarghya Ghose1, Krishan Gopal Thakur1 and Dipak Dutta1*

7 CSIR-Institute of Microbial Technology, Chandigarh, India1

10 * Corresponding author

11 E-mail: [email protected] (DD)

25 1

26 Abstract

27 The nick translation property of DNA polymerase I (Pol I) ensures the maturation of

28 Okazaki fragments by removing primer RNAs and facilitating ligation. However, prolonged

29 nick translation traversing downstream DNA is an energy wasting futile process, as Pol I

30 simultaneously polymerizes and depolymerizes at the nick sites utilizing energy-rich dNTPs.

31 Using an in vitro assay system, we demonstrate that the -clamp of the Escherichia coli

32 replisome strongly inhibits nick translation on the DNA substrate. To do so, -clamp inhibits

33 the strand displacement activity of Pol I by interfering with the interaction between the finger

34 subdomain of Pol I and the downstream primer-template junction. Conversely, -clamp

35 stimulates the 5’ exonuclease property of Pol I to cleave single nucleotides or shorter

36 oligonucleotide flaps. This single nucleotide flap removal at high frequency increases the

37 probability of ligation between the upstream and downstream DNA strands at an early phase,

38 terminating nick translation. Besides -clamp-mediated ligation helps DNA ligase to seal the

39 nick promptly during the maturation of Okazaki fragments.

41 Introduction

42 In replicating E. coli cells, DNA polymerase III (Pol III) holoenzyme is the major

43 replicative DNA polymerase [1]. Three Pol III core complexes (each containing 

44 polypeptides) interact with the clamp loader complex (12’ polypeptides) and -sliding

45 clamps at the primer-template junctions of a replication fork [2-8]. The dimeric -clamp is a

46 doughnut-shaped molecule that accommodates double-stranded (ds) DNA at the central hole

47 [2]. When β is free in solution, ATP-bound form of clamp loader complex drives β-clamp

48 opening and binding to the single-stranded (ss) DNA region of a primer-template junction [9-

49 12]. Such a clamp loader interaction with the DNA triggers ATP hydrolysis and results in β-

50 clamp loading on the primer-template junction. Subsequently, the clamp loader affinity to the

51 β-clamp and DNA decreases rapidly that allows clamp loader to dissociate [13,14].

52 Immediately after being loaded onto the DNA, the sliding clamp interacts with Pol III core at

53 the replication fork [15-17]. The -clamp also has affinity for all other DNA polymerases in

54 E. coli [18-25]. These interactions dramatically increase the processivity of the DNA

55 polymerases [18-25]. Furthermore, interactions of -clamp with DNA ligase and MutS repair

56 proteins have also been reported [19]. Therefore, apart from acting as a processivity factor for

57 the replicating DNA polymerases, the -clamp may have some other putative roles in the

58 post-replication DNA repair processes.

59 Synthesis of the leading strand DNA presumably requires a few priming events [26-

60 30], while frequent priming events at the lagging strand generate discrete Okazaki fragments

61 of 1-2kb in length [31,32]. Maturation of the Okazaki fragments eventually produces an

62 intact lagging strand primarily by the nick translating DNA polymerase I (Pol I) and ligase

63 functions. Pol I is a multifunctional protein with a large and a small domains connected by a

64 flexible linker [33,34]. During nick translation, the large domain of Pol I (also known as

65 Klenow fragment) polymerizes from the 3’ end at the nick and simultaneously displaces 5’

66 end nucleotides from the template to create variable lengths of oligonucleotide flaps at the

67 nick site [33-37]. Concomitantly, the small domain of Pol I excises the flap by its flap

68 endonuclease activity [33-37]. The strand displacement and flap endonuclease activities are

69 together responsible for the 5’ to 3’ exonuclease (hereinafter 5’ exonuclease) function of Pol

70 I. The Klenow fragment of Pol I also exhibits 3’to 5’ exonuclease (hereinafter 3’

71 exonuclease) function that is responsible for the proofreading activity [33].

72 After synthesizing an Okazaki fragment, the replisome dissociates from the -clamp.

73 As a result, the “left over” -clamp remains attached at the 3’-side of the ssDNA gap or nick 3

74 [38-41]. Consistently, -clamp has been shown to dynamically accumulate behind

75 progressing replication fork in E. coli and Bacillus subtilis [42,43]. Presumably, -clamp

76 subsequently helps in repairing the gap or nick by interacting with Pol I, and DNA ligase

77 [19,44,45]. Although a growing body of in vitro studies in the past have been done to

78 illuminate the different functions of Pol I, as described above [33-37,46], none of them

79 addressed the dynamics and biochemistry of these functions in the presence of -clamp.

80 Here, we designed in vitro experiments to assess the influence of -clamp on the functions of

81 Pol I and DNA ligase in the nick translation process. Our approach unraveled a hitherto

82 unknown mechanism of clamp-assisted DNA gap repair in E. coli.

83 Results

84 -clamp inhibits Pol I-mediated nick translation

85 To probe the effects of -clamp on nick translation process, we assembled a template

86 annealing three different oligonucleotides (67, 19 and 28 bases long), as described in

87 Materials and Methods section. Thus, as depicted in the Fig 1A, we created a template that

88 has a primed template junction with a 3’ recessed end of 19 bases long primer and 20

89 nucleotides single-stranded gap, which suitably acts as a “placeholder” to loaded- blocking

90 its free sliding [12]. The efficiency and accuracy of primer annealing were almost 100%, as

91 judged by native PAGE (S1 Fig). We loaded -clamp on the template with the help of clamp

92 loader and ATP, and initiated the polymerization reaction by adding Pol I and dNTPs. The

93 pattern of radioactive oligonucleotide bands in the denaturing gel revealed that Pol I filled the

94 20 nucleotides DNA gap by extending the radiolabeled 19 bases upstream oligonucleotide

95 and encountered the 28 bases downstream RNA oligonucleotide to initiate nick translation

96 (Fig 1B). Interestingly, -clamp slightly stimulated pausing of the nick translating Pol I

97 scattered between 39th to 67th positions while traversing 28 bases downstream RNA

98 oligonucleotide (compare the dotted lines in lane 2 and lane 6 of Fig 1B), indicating that -

99 clamp could moderately reduce the speed of nick translation. On calculating the rate of nick

100 translation, we show that the effect of -clamp on the Pol I dependent nick translation

101 traversing RNA substrate was nominal (S2 Fig). However, when we used the template with

102 the 28 bases downstream DNA oligonucleotide, the -clamp dramatically increased the pause

103 of nick translating Pol I, as judged by the bulk migration of the nick sites at 30 and 60

104 seconds time span (Fig 1C; zones II and I).

105

106 Fig 1. β-clamp inhibits Pol I-mediated nick translation. (A) The template used in the assay

107 was prepared by assembling 67 bases, 5’-phosphorylated 28 bases and 5’-radiolabelled

108 (asterisk) 19 bases oligonucleotides. (B) A representative urea denaturing PAGE and the rate

109 calculation therein (see also S2 Fig) indicate that the presence of β-clamp nominally affected

110 nick translation traversing 28 bases downstream RNA. (C) The urea-denaturing gel shows

111 that Pol I efficiently translated the nick, degrading 28 bases downstream DNA, but the

112 presence of β-clamp dramatically reduced the speed of nick translation. The presence of

113 ligase allowed early ligation when β-clamp slowed down Pol I-mediated nick translation. (D)

114 The urea PAGE represents that the 3’-exo- Pol I-mediated nick translation was slowed down

115 in the presence of template-loaded β-clamp, in a manner similar to the nick translation with

116 β-clamp and Pol I combinations, as shown in panel C. Moreover, the presence of template-

117 loaded β-clamp increases the ligation efficiency during the 3’-exo- Pol I-mediated nick

118 translation. (E) The urea denaturing gel represents that the 5’-exo- Pol I intensely paused at

119 the 39th nucleotide and downstream positions, affecting the ligation process. The presence of

120 template-loaded β-clamp moderately enhances these pauses, but facilitates ligation to some

121 extent. The # indicates the position of truncated by-products that originated from the 67- 5

122 nucleotide long product by the 3’ exonuclease function of Pol I and 5’-exo- Pol I (panel B, C,

123 E). This truncated product is missing in the assay with 3’-exo- Pol I (panel D). All the

124 experiments were performed at least three times. The values represent mean ± standard

125 deviation (sd).

126

127 We conducted the control experiments to show that the clamp loader could efficiently

128 load -clamp on the above-mentioned specific template types (see the detail in S1 text). For

129 this, we used biotinylated templates or histidine-tag version of -clamp to show that after a

130 clamp loading reaction, the template or the -clamp could pull-down -clamp or template,

131 respectively, with 100% occupancy, while clamp loader proteins were washed away (S3 Fig).

132 This observation suggests that the clamp loading was efficient that allows a stable -clamp

133 interaction with the DNA containing the primer-template junction. Further, to show that the

134 observed effect -clamp on nick translation is not an artifact arisen by an interference of

135 clamp loader proteins, we performed clamp-loading reaction and washed the streptavidin-

136 bound biotinylated template to remove the clamp loader proteins. Next, we added Pol I and

137 dNTPs to show that the template-bound  could inhibit nick translation similar to the

138 unwashed control reaction containing clamp loader proteins (S4Fig). This data suggests that

139 the template-loaded -clamp, but not the free clamp loader proteins, in the reaction mixture

140 interfere with the nick translation by Pol I. These observations are consistent with the original

141 literature, which have demonstrated that -clamp does not slide freely from a template that

142 has a primer-template junction, and the affinity of clamp loader proteins to -clamp declines

143 rapidly after latter is loaded on the template [9,47].

144 We incorporated point mutations in polA (the Pol I encoding gene) by site directed

145 mutagenesis to disrupt the 3’ and 5’ exonuclease activities. The purified 3’-exonuclease-free

146 (3’-exo-) and 5’-exonuclease-free (5’-exo-) Pol I were then used to dissect the role of 3’ and

147 5’ exonuclease activities of Pol I in the nick translation process. The nick translation

148 mediated by the 3’-exo- Pol I (Fig 1D) appeared to be faster than the Pol I-mediated nick

149 translation (compare the intensity of paused bands and 67-nucleotide product between lane 2

150 of Fig 1C and Fig 1D respectively). To further elaborate this observation, in a separate

151 experiment, we determined that the rate of nick translation mediated by 3’ exo- Pol is faster

152 than the Pol I-mediated nick translation (S5 Fig). However, in the presence of the template-

153 loaded -clamp, 3’-exo- Pol I-mediated nick translation (Fig 1D) was found to be broadly

154 similar to the Pol I-mediated nick translation patterns (compare lanes 7 and 8 between Fig 1C

155 and Fig 1D). This observation indicates that the 3’-exonuclease activity of Pol I has little

156 impact on the speed of nick translation in the presence of template-loaded -clamp. On the

157 other hand, the 5’-exo- Pol I exhibited intense pauses at the 39th nucleotide and few bases

158 downstream positions (dotted zone at lanes 2 and 3; Fig 1E). The template-loaded -clamp

159 modestly enhances the intensity of 5’-exo- Pol I pauses (dotted zone at lanes 7 and 8; Fig 1E).

160 These observations indicate that the elimination of 3’-exonuclease and 5’-exonuclease

161 activities impair the coordination in the strand displacement synthesis process, leading to

162 increased and decreased speed of nick translation, respectively, by Pol I.

163 -clamp inhibits strand displacement function of Pol I

164 The Klenow fragment of Pol I exhibits polymerization, 3’exonuclease and 5’ to 3’

165 strand displacement activity [33,34]. To determine if the slow nick translation by clamp-

166 bound Pol I through the DNA substrate was the result of impaired strand displacement, we

167 replaced E. coli Pol I with the exonuclease-free Klenow (exo- Klenow), which lacks both 3’

168 and 5’ exonuclease functions, in the assay. The exo- Klenow exhibited a trail of scattered

169 pause complexes, as judged by the band intensities between the 39th to 67th nucleotide

170 positions (dotted zone at lanes 2 and 3; Fig 2A). Remarkably, the presence of template-loaded

171 -clamp further intensified the pausing of exo- Klenow, mostly at the 39th nucleotide position

172 (zone III; Fig 2A) where the nick translation begins.

173

174 Fig 2. β-clamp inhibits strand displacement activity of exo- Klenow and exo- Pol I

175 (A) The urea PAGE shows that exo- Klenow exhibits a slower speed of strand displacement.

176 Presence of the template-loaded β-clamp stalls the exo- Klenow pauses mostly at the 39th

177 nucleotide position. The exo- Klenow-mediated strand displacement coupled with ligation

178 represents that the DNA ligase fails to ligate nicks. However, the presence of template-loaded

179 β-clamp increases the ligation frequency. (B) exo- Pol I exhibits similar functional

180 consequences on the strand displacement and ligation as observed in case of exo- Klenow

181 (panel A). The values representing mean ± sd are calculated from three different experiments.

182

183 The choice of exo- Klenow of Pol I, in which the entire small domain containing 5’-

184 exonuclease activity is missing could be non-ideal, because the observed effects might be due

185 to a conformational change rather than stemming from the loss of 5’-exonuclease activity. To

186 test this issue, we generated a mutated version of Pol I, which encodes 3’- and 5’-

187 exonuclease-free Pol I (exo- Pol I) but retains both of the domains. Using the exo- Pol I, we

188 show that the pattern of paused complexes (dotted zones at the lanes 2 and 3; Fig 2B) was

189 comparable to the paused complexes observed with the exo- Klenow (Fig 2A) in both the

190 absence and presence of the template loaded -clamp. This data indicates that the elimination

191 of 3’ and 5’-exonuclease activities, but not the deletion of small domain, induce the observed

192 paused complexes in the absence or presence of the template loaded -clamp. As visually

193 observed, exo- pol I was apparently more efficient in producing 67 nucleotides long product

194 than exo- Klenow in the absence of template loaded β-clamp (Fig 2A and 2B). 8

195 -clamp promotes ligation at an early stage of nick-translation

196 Apart from Pol I, DNA ligase also interacts with the -clamp [19]. Therefore, the

197 polymerization and flap endonuclease activities of Pol I, which fills the ssDNA gap to

198 produce a nick and continuously generates new 5’ ends during nick translation, respectively,

199 may facilitate ligation between the upstream and downstream DNA strands. To probe this

200 working hypothesis, we performed an assay coupled with E. coli DNA ligase. We observed

201 that ligase along with Pol I, both in the presence or absence of the template-loaded -clamp,

202 produced more intense 67 bases long final product than Pol I alone produced in 30 seconds of

203 reaction span (Fig 1C). This observation indicates that ligation by the ligase action during

204 ongoing nick translation quickly contributes creating a fraction of the 67 bases long products.

205 Therefore, almost all of the scattered nick t