Repair of protein-linked DNA double strand breaks: Using the adenovirus genome as a model substrate in cell-based assays

Abstract

The DNA double strand breaks (DSBs) created during meiotic recombination and during some types of chemotherapy contain protein covalently attached to their 5' termini. Removal of the end-blocking protein is a prerequisite to DSB processing by non-homologous end-joining or homologous recombination. One mechanism for removing the protein involves CtIP-stimulated Mre11-catalyzed nicking of the protein-linked strand distal to the DSB terminus, releasing the end-blocking protein while it remains covalently attached to an oligonucleotide. Much of what is known about this repair process has recently been deciphered through in vitro reconstitution studies. We present here a novel model system based on adenovirus (Ad), which contains the Ad terminal protein covalently linked to the 5' terminus of its dsDNA genome, for studying the repair of 5' protein-linked DSBs in vivo. It was previously shown that the genome of Ad mutants that lack early region 4 (E4) can be joined into concatemers in vivo, suggesting that the Ad terminal protein had been removed from the genome termini prior to ligation. Here we show that during infection with the E4-deleted Ad mutant dl1004, the Ad terminal protein is removed in a manner that recapitulates removal of end-blocking proteins from cellular DSBs. In addition to displaying a dependence on CtIP, and Mre11 acting as the endonuclease, the protein-linked oligonucleotides that are released from the viral genome are similar in size to the oligos that remain attached to Spo11 and Top2 after they are removed from the 5' termini of DSBs during meiotic recombination and etoposide chemotherapy, respectively. The single nucleotide resolution that is possible with this assay, combined with the single sequence context in which the lesion is presented, make it a useful tool for further refining our mechanistic understanding of how blocking proteins are removed from the 5' termini of DSBs.

Keywords: Adenovirus; Chemotherapy; Concatemer; CtIP; DNA double strand break; End blocking; End processing; Endonuclease; Etoposide; Homologous recombination; Ligation; MRN; Mre11; Nonhomologous end joining; Protein-DNA adduct; Protein-linked DSB; Spo11; Terminal protein; Topoisomerase 2; dl1004.

Fig. 1.

Current understanding of 5′ PL-DSB processing. When covalently trapped on the 5′ termini of a DSB, Top2 protomers can be digested by the proteasome. The resultant peptides, which remain covalently bound to the 5′ termini of the DSB, can subsequently be removed by the TDP2 phosphodiesterase to generate “clean” ends. Alternatively, Top2 can be removed via an endonucleolytic mechanism wherein the released intact protomers remain covalently attached to an oligonucleotide. Data suggest that Mre11 acts as the endonuclease and is stimulated by CtIP. Prot., proteasome.

Fig. 2.

Protein priming of Ad genome replication. The 5′ termini of the linear double stranded Ad genome are perpetually linked to Ad TP via a serine phosphodiester bond. A stable heterodimer composed of the Ad DNA Polymerase (Pol) and Ad TP specifically binds the termini of the viral genome. Here the complex then binds dCTP, positioning it opposite to the 3′ terminal guanine on the templating strand. Attack of the α-phosphate of this nascent dCTP by a serine hydroxyl in TP results in a serine-dCMP covalent adduct. It is the S′-hydroxyl of this serine-dCMP moiety that Ad DNA Pol utilizes as a primer. Standard strand displacement DNA synthesis (in the absence of lagging strand synthesis) generates one intact duplex molecule and one single stranded molecule. The latter can be utilized as a template in a subsequent round of replication. Alternatively, when replication is initiated at both termini of a double stranded Ad genome, two duplex daughter molecules are produced simultaneously. Importantly, the sequence of the first 103 nucleotides at each terminus of the Ad serotype 5 (Ad5) genome are identical to one another, suggesting that the nuances of replication initiation should be conserved at each terminus (i.e. differences associated with sequence context should be negated). The 5′ terminal sequences of WT Ad5 are conserved in the dl1004 mutant.

Fig. 3.

Detection of covalent TP-oligonucleotide complexes during dl1004 infection. (A) Schematic of labeling assay. TdT, terminal deoxynucleotidyl transferase. (B) TP immunoprecipitated from dl1004-infected HeLa, U2OS, or HFFF cells can be labeled with [α-³²P]-cordycepin triphosphate by TdT. The arrow indicates the location of “free” TP, as determined by immunoblotting. DBP, Ad DNA binding protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (C) Testing the presumed “TP-oligo” species for protein, DNA, and a serine phosphodiester linkage using Proteinase K, mung bean (MB) nuclease, or piperidine, respectively. (D) The mobility differences of the virus-specific, TdT-dependent cluster of TP bands are not due to differential phosphorylation. CIP, calf intestinal phosphatase; Na₃VO₄, the phosphatase inhibitor sodium orthovanadate; RPA32, the 32 kDa subunit of replication protein A; RPA32 (S4/S8), RPA phosphorylated at serines 4 and 8. (E) 2D gel analyses of TP-oligo from mock- and dl1004-infected HeLa cells. Isoelectric focusing is along the x-axis while conventional SDS-PAGE is along the y-axis. (F) Analysis of labeled DNA oligos that were released from immunoprecipitated TP by incubation in piperidine, by separation on a DNA sequencing gel. Numbers denote the location of reference oligonucleotides derived from the 5′ termini of the Ad dl1004 genome (which are identical to one another for the first 103 nucleotides).

Fig. 4.

Variation in TP-oligo size during dl1004 vs. WT Ad5 infection. HeLa cells were infected with dl1004 (A) or WT (B), harvested at the indicated time post infection, and TP was immunoprecipitated with the indicated antibody before being labeled with [α-³²P]-cordycepin triphosphate by TdT. Nbs1, Nijmegen breakage syndrome protein 1 (Nibrin); DBP, Ad DNA binding protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; h.p.i., hours post infection.

Fig. 5.

TP-oligo production during dl1004 vs. WT Ad5 infection of HeLa cells. (A) TP immunoprecipitated from WT-infected HeLa cells and then labeled with [α-³²P]-cordycepin triphosphate by TdT displays a lower apparent MW than that derived from dl1004-infected cells. DBP, Ad DNA binding protein. (B) On a 2D gel TP-oligo derived from WT infected cells consists of fewer species than what is seen in dl1004-infected cells. Dotted lines help position the two samples relative to one another, and highlight differences in banding patterns. (C) Comparison of TP-linked oligonucleotides, after release from TP via piperidine-catalyzed hydrolysis, from WT- and dl1004-infected HeLa cells. (D) Analysis of the size and distribution of TP-oligo species during dl1004 or WT infection of a HeLa(shNbs1) cell line. (E) DNA sequencing gel analysis of the same samples from “D”, after cleaving the labeled DNA oligos from TP by incubation in piperidine. In both “C” and “E” the numbers denote the location of reference oligonucleotides derived from the 5′ termini of the Ad genome (which are identical to one another for the first 103 nucleotides; this sequence is the same in both dl1004 and WT Ad5).

Fig. 6.

Role of Mre11 and CtIP in TP-oligo generation. (A) When either Mre11 or CtIP are knocked down, the amount of TP-oligo is reduced and only the lowest MW species are present. (B) Knockdown of BRCA1 has no impact on either the abundance or the size distribution of the TP-oligo species. (C) Simultaneous knockdown of Mre11 and CtIP is not more deleterious to TP-oligo production than knockdown of either gene alone. (D) Expression of shRNA resistant forms of WT Mre11 or WT CtIP restores TP-oligo production (both the quantity and the size distribution) within knockdown cell lines. In contrast, add back of the nuclease dead Mre11-3 mutant does not rescue production of the high molecular weight TP oligo species.