Bacterial Nonhomologous End Joining Ligases Preferentially Seal Breaks with a 3′-OH Monoribonucleotide

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 alkalilabile 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.Direct evidence that bacteria catalyze DNA double-strand break (DSB) 2 repair via nonhomologous end joining (NHEJ) emerged from studies of the repair of linear plasmid DNAs transfected into mycobacteria (1). NHEJ entails approximation of the broken DNA ends, aided by the bacterial end-binding protein Ku, followed by sealing of at least one of the broken strands by a specialized bacterial ATP-dependent DNA ligase, either LigD or LigC (1-9, 36). Unlike homologous recombination, which is generally error-free, NHEJ can be either faithful or mutagenic. The signature feature of NHEJ in mycobacteria is that half of the repair events at blunt or complementary 5Ј-overhang DSBs are unfaithful because the DSB ends are extended by polymerases or resected by nucleases prior to sealing by ligase (1,8,9,36). Because NHEJ does not rely on a homologous DNA template, it can operate when only one chromosomal copy is available, e.g. during late stationary phase or after sporulation (10 -13). Whereas mutagenic DSB repair might seem counterproductive, it can be advantageous, especially if the alternative is death of the quiescent bacterium or spore.Biochemical, structural, and genetic studies of the NHEJ ligases and Ku proteins of Mycobacterium, Pseudomonas, Bacillus, and Agrobacterium are beginning to define a pathway with unique features and distinctive enzymatic components (1, 4 -9, 14 -17, 36). Ku and LigD are critical agents of the pathway. The efficiency of plasmid-based NHEJ of blunt and 5Ј-overhang DSBs in mycobacteria is reduced several hundredfold by deletion of Ku and by ϳ100-fold by deletion of LigD (1, 36). LigC provides an efficient backup sealing function in mycobacteria when LigD sealing activity is a...

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

“…2, 14 and results of this study). Thus some rNMPs are likely to be stably incorporated into DNA during replication, and possibly during DNA repair, e.g., nonhomologous end joining (NHEJ) of double strand breaks in DNA (9,15). This possibility is supported by biochemical studies implicating RNase H2 and FEN1 in the repair of single ribonucleotides in DNA (16,17).…”

“…For example, DNA polymerase μ incorporates rNMPs into DNA to such an extent that it is suggested to use rNTPs as normal precursors during NHEJ of double strand DNA breaks in the G1 phase of the cell cycle, when dNTP concentrations are particularly low (9). Here it may be relevant that ligases involved in NHEJ prefer to seal strand breaks containing a monoribonucleotide on the 3′-OH end (9,15). Extending this logic, it is theoretically possible that rNMPs may be incorporated into DNA during repair synthesis performed by either Pol δ or Pol ε, e.g., during mismatch repair, nucleotide excision repair, or break-induced recombination.…”

Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates

“…In many bacteria, LigD is found as a fusion of DNA ligase (Lig), polymerase (Pol), and phosphoesterase (PE) domains in a variety of configurations. The NHEJ Lig or ligase domain (LigDom) has a strong propensity for ligating nicks with a ribonucleotide on the 3′ position of the break (12), and the Pol domain (PolDom) preferentially incorporates nucleoside triphosphates (NTPs), rather than the expected deoxynucleoside triphosphates (dNTPs) (12,13). Together, these findings establish that NHEJ repair requires Pol insertion of ribonucleotides at the DNA termini for efficient ligation.…”

Cost of rNTP/dNTP pool imbalance at the replication fork

“…For example, eukaryotic Pol mu is highly proficient at rNTP incorporation and is suggested to use rNTPs during nonhomologous end joining of ds breaks at low dNTP concentration (37). Indeed, it appears that ligases involved in nonhomologous end joining are more proficient in using a 3′ terminal rNMP (38,39). Moreover, in S. pombe, two rNMPs are incorporated in S phase and retained during the next round of replication, which enhances the mating type switch recombination event (39).…”