Abstract
Many bacterial species have a nonhomologous end joining system of DNA repair driven by dedicated DNA ligases (LigD and LigC). LigD is a multifunctional enzyme composed of a ligase domain fused to two other catalytic modules: a polymerase that preferentially adds ribonucleotides to double-strand break ends and a phosphoesterase that trims 3'-oligoribonucleotide tracts until only a single 3'-ribonucleotide remains. LigD and LigC have a feeble capacity to seal 3'-OH/5'-PO(4) DNA nicks. Here, we report that nick sealing by LigD and LigC enzymes is stimulated by the presence of a single ribonucleotide at the broken 3'-OH end. The ribonucleotide effect on LigD and LigC is specific for the 3'-terminal nucleotide and is either diminished or abolished when additional vicinal ribonucleotides are present. No such 3'-ribonucleotide effect is observed for bacterial LigA or Chlorella virus ligase. We found that in vitro repair of a double-strand break by Pseudomonas LigD requires the polymerase module and results in incorporation of an alkali-labile ribonucleotide at the repair junction. These results illuminate an underlying logic for the domain organization of LigD, whereby the polymerase and phosphoesterase domains can heal the broken 3'-end to generate the monoribonucleotide terminus favored by the nonhomologous end joining ligases.
Highlights
Ing by ligase [1, 8, 9, 36]
To gauge whether LigD activity was affected by ribonucleotides on the 3Ј-OH strand, we compared a series of singly nicked 24-bp duplexes (5Ј-32Plabeled at the nick) in which the unlabeled 12-mer 3Ј-OH strand consisted of 12 deoxynucleotides (D12), 11 deoxynucleotides and 1 ribonucleotide (D11R1), 10 deoxynucleotides and 2 ribonucleotides (D10R2), or 9 deoxynucleotides and 3 ribonucleotides (D9R3)
LigA and Chlorella virus ligase were unaf- characterized to date
Summary
Templated ribonucleotide addition is limited to about four cycles of rNMP incorporation because the primer-template is rendered progressively less active as ribonucleotides accumulate at the 3Ј-end [14] These properties suggest that the initial insertions preceding the sealing step of NHEJ might involve rNMP incorporation. The phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5Ј-single-strand tail of the primer-template substrate These properties distinguish LigD PE from other 3Ј-end-processing enzymes. We found that strand sealing is stimulated by a single ribonucleotide at the 3Ј-OH terminus of the break This 3Ј-ribonucleotide dependence is a signature property of NHEJ ligases (not shared with bacterial LigA or a prototypal eukaryotic ATP-dependent ligase) that rationalizes the bundling of auxiliary catalytic modules within LigD enzymes.
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