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Many biological scenarios generate "dirty" DNA 3'-PO4 ends that cannot be sealed by classic DNA ligases or extended by DNA polymerases. The noncanonical ligase RtcB can "cap" these ends via a unique chemical mechanism entailing transfer of GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form DNA3'pp5'G. Here, we show that capping protects DNA 3' ends from resection by Escherichia coli exonucleases I and III and from end-healing by T4 polynucleotide 3' phosphatase. By contrast, the cap is an effective primer for DNA synthesis. E. coli DNA polymerase I and Mycobacterium DinB1 extend the DNAppG primer to form an alkali-labile DNApp(rG)pDNA product. The addition of dNTP depends on pairing of the cap guanine with an opposing cytosine in the template strand. Aprataxin, an enzyme implicated in repair of A5'pp5'DNA ends formed during abortive ligation by classic ligases, is highly effective as a DNA 3' decapping enzyme, converting DNAppG to DNA3'p and GMP. We conclude that the biochemical impact of DNA capping is to prevent resection and healing of a 3'-PO4 end, while permitting DNA synthesis, at the price of embedding a ribonucleotide and a pyrophosphate linkage in the repaired strand. Aprataxin affords a means to counter the impact of DNA capping.

The central dogma of DNA enzymology has long held that for two pieces of DNA to be ligated, the respective DNA pieces must bear a hydroxyl on the 3′-end and a phosphate on the 5′-end. All of the other types of DNA ends are considered incorrect or “dirty,” as they cannot be joined by classic DNA ligases. However, intrinsic and extrinsic DNA damage often results in broken DNA with dirty ends. Thus, there are a variety of DNA repair enzymes that function to “clean up” or “heal” such dirty ends to promote the restoration of a continuous DNA phosphodiester backbone. Until now, DNA breaks with phosphate at the 3′-end and hydroxyl at the 5′-end also fell into the category of dirty ends. The study in PNAS by Das et al. (1) demonstrates that a recently identified RNA-processing enzyme, RtcB, can directly ligate a DNA 3′-phosphate end with a DNA 5′-OH end (Fig. 1). Fig. 1. Comparison of the end-joining mechanisms between classic ligases ( A ) and RtcB ( B ). ( A ) First, the catalytic lysine residues of classic DNA and RNA ligases are charged by adenylylation. Second, the transfer of adenylate to the 5′-phosphate end of DNA or RNA activates the 5′-end with a pyrophosphate linkage. Third, the attack by the 3′-OH on the 5′-phosphoanhydride generates the 3′-5′ phosphodiester bond, releasing AMP. ( B ) In a GTP-dependent step, RtcB first becomes charged by guanylylation at a conserved histidine residue located at the catalytic site. Next, the transfer of guanylate from the histidine residue to the RNA or DNA 3′-phosphate end generates … [↵][1]1To whom correspondence should be addressed. E-mail: pommier{at}nih.gov. [1]: #xref-corresp-1-1

Significance The ability to repair breaks in the DNA phosphodiester backbone is essential for genome integrity. When breakage results in 5′-PO 4 and 3′-OH termini, the ends can be rejoined to each other, or to novel partner strands, by classic DNA ligases that covalently activate the 5′-PO 4 end by linkage to AMP. However, when breakage leaves 5′-OH and 3′-PO 4 termini, the ends are considered “dirty” because they cannot be sealed by classic ligases. This paper shows that the unconventional ligase RtcB can evade the DNA dirty end chemistry problem by splicing DNA 3′-PO 4 ends to DNA 5′-OH ends. RtcB accomplishes this by attaching a GMP nucleotide to the DNA 3′-PO 4 end to activate it for nucleophilic attack by the 5′-OH.

Intracellular forms of pseudorabies virus parental DNA were examined before and after the onset of viral DNA synthesis. Before initiation of synthesis, parental viral DNA acquires single-stranded ends. Circular and concatemeric molecules are also observed, indicating that the single-stranded ends are complementary. Viral DNA replication is initiated at an internal site within the DNA molecule, giving rise to characteristic replicative loops with single-stranded regions in the trans position. Such replicative loops were seen in unit-size (and smaller than unit-size) linear molecules as well as in circular and concatemeric molecules. These results show that the parental viral DNA molecules that acquire single-stranded ends, and consequently are able to form circles and concatemers, proceed to replicate.

In animal cells, exogenous DNA recombines into random chromosomal sites much more frequently than it recombines into homologous sites. Free DNA ends are "recombinogenic" in both processes. To test the effects of specific ends on analogous extrachromosomal processes, we constructed a linear genome of simian virus 40 with terminal repeated sequences. After transfection into monkey cells, the model substrate can circularize by end joining (analogous to random integration) or by homologous recombination between its terminal repeats (analogous to targeted recombination). Since the two types of recombination are in competition with one another, the ratio of homologous-recombination to end-join products is a sensitive indicator of the differential effects of specific ends. Substrates with blunt ends, complementary sticky ends, or mismatched ends generated the same ratio of homologous-recombination to end-join products. However, addition of dideoxynucleotides to the 3' hydroxyls of the substrate decreased the frequency of end joining by a factor of 5-6 relative to homologous recombination. Thus, the frequency of end joining can be decreased relative to that of homologous recombination by modification of the ends of the input DNA. These results suggest an approach to altering the ratio of random to targeted integration in mammalian cells.

The ring closure probability, or j factor, has been measured for DNA restriction fragments of defined sequence bearing EcoRI cohesive ends and ranging in size from 126 to 4361 base pairs (bp). The j factor is defined as the ratio of the equilibrium constants for cyclization and for bimolecular association via the cohesive ends. The end-joining reactions are fast compared to covalent closure of the cohesive ends by T4 DNA ligase. The rate of ligase closure is shown to be proportional to the equilibrium fraction of DNA molecules with joined cohesive ends, both in cyclization and in bimolecular association reactions. The j factor changes by less than 10-fold between 242 and 4361 bp, whereas it decreases by more than 100-fold between 242 and 126 bp as the DNA reaches the size range of the persistence length (150 bp). As regards ring closure, short DNA fragments are surprisingly flexible. These data are in good agreement with predictions by others for the ring closure probability of a wormlike chain.

Reversed phase column chromatography on RPC-5 resin was used to fractionate milligram quantities of DNA fragments generated by restriction endonucleases. Fractionation was on the basis of size, the presence or absence of sticky ends, and at least one as yet undertermined property.

DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.

Based on the 3′- and 5′-terminal sequences of DNA of phage λ, P2, and 186, a model is proposed for recognition of DNA sequences by enzymes responsible for generation of cohesive ends. Two copies of the cohered ends, either on separate molecules or on a concatemer, are aligned with their helical axes parallel but running in opposite directions. The nicking system is dimeric, with each of the two monomers carrying identical sequence-recognition sites. Two pairs of nicks are introduced into the two aligned DNA molecules by the nicking system. The applicability of this model to other biological processes, such as integration of a viral genome into a host genome and the cutting of concatemeric T7 DNA, is discussed.

Linear double-stranded molecules of the circularly permuted and terminally redundant DNA of Salmonella bacteriophage P22 have been converted to oligomeric products in the presence of polynucleotide ligase coded for by the coliphage T4. The reaction has been monitored by sucrose density-gradient centrifugation and electron microscopy. It goes slowly and gives yields of 30-40%. The products are mainly dimers and trimers, but higher oligomers are also present. DNA ligase extracted from uninfected Escherichia coli seems unable to perform a similar reaction, which is concluded to involve the fully base-paired termini. Linear double-stranded molecules of simian virus(SV) 40 DNA, produced by the action of the bacterial restriction endonuclease R 1 , are oligomerized by either ligase; therefore, this reaction seems to involve single-stranded cohesive ends. No mixed products could be found when P22 DNA and linear SV 40 DNA were exposed together to the T4 ligase.