Poly(ADP-Ribose) Polymerase 2 Recruits Replication Protein A to Sites of LINE-1 Integration to Facilitate Retrotransposition

Abstract

Long interspersed element-1 (LINE-1 or L1) retrotransposition poses a threat to genome integrity, and cells have evolved mechanisms to restrict retrotransposition. However, how cellular proteins facilitate L1 retrotransposition requires elucidation. Here, we demonstrate that single-strand DNA breaks induced by the L1 endonuclease trigger the recruitment of poly(ADP-ribose) polymerase 2 (PARP2) to L1 integration sites and that PARP2 activation leads to the subsequent recruitment of the replication protein A (RPA) complex to facilitate retrotransposition. We further demonstrate that RPA directly binds activated PARP2 through poly(ADP-ribosyl)ation and can protect single-strand L1 integration intermediates from APOBEC3-mediated cytidine deamination in vitro. Paradoxically, we provide evidence that RPA can guide APOBEC3A, and perhaps other APOBEC3 proteins, to sites of L1 integration. Thus, the interplay of L1-encoded and evolutionarily conserved cellular proteins is required for efficient retrotransposition; however, these interactions also may be exploited to restrict L1 retrotransposition in the human genome.(A) The L1.3 ORF2p-3FLAG expression vector (pTMO2F3). The CMV promoter (black arrow) and 5 0 UTR augment L1 expression. Also indicated are the FLAG epitope tag, SV40 polyadenylation signal, and the EN, Z, RT, and cysteine-rich (C) domains. (B) The ORF2p-3FLAG complex. HEK293T cells were transfected with pAD500 (ORF2p-TAP) or pTMO2F3 (ORF2p-3FLAG). Left: the ORF2p-3FLAG complex was purified using anti-FLAG antibody-conjugated beads, separated on a denaturing polyacrylamide gel, and visualized by silver staining. Right: ORF2p-3FLAG was detected by western blot. Input, the whole-cell lysate used in the IP. Arrows, ORF2p-3FLAG. (C) Validation of proteins in the ORF2p-3FLAG complex. ORF2p-3FLAG complexes were purified (see B) from HEK293T cells transfected with a full-length (pTMF3, lane 2) or monocistronic (pTMO2F3, lane 4) L1 expression constructs. The ORF1p-1FLAG complex was purified from cells transfected with pJM101/L1.3FLAG (lane 5). The pJM101/L1.3 and pAD001 samples (lanes 1 and 3, respectively) served as negative controls. Input, western blot lanes of whole-cell lysate used in the IP. Red triangles, FLAG tags; SSB, single-strand break; NHEJ, non-homologous end-joining; NER, nucleotide excision repair. See also Figure S1 and Table S1.

Replication protein A: a multifunctional protein with roles in DNA replication, repair and beyond

“…This triggers the recruitment of RPA at L1 integration sites to facilitate retrotransposition. Interestingly, RPA can also guide the cytidine deaminase APOBEC3A to sites of L1 integration ( 254 ) that can generate a cytosine to thymine mutation. This is reminiscent of previous studies, which reported that RPA can interact with the AID to mediate somatic hypermutation and class switch recombination of immunoglobulin genes ( 46 , 255 ).…”

Activation of PARP2/ARTD2 by DNA damage induces conformational changes relieving enzyme autoinhibition

“…In the nucleus, the main enzymes carrying out poly-ADP-ribosylation are poly(ADP-ribose) polymerase 1 (PARP1) and PARP2. These enzymes bind to the damaged DNA and subsequently generate poly-ADP-ribose (PAR) chains that act as recruitment signals for a range of DNA repair factors 4 – 6 . The role of poly-ADP-ribosylation is established in single-strand break repair (SSBR) and in alternative non-homologous end joining (aNHEJ) mechanisms, where the key proteins involved in the repair pathways are known to be recruited to the site of DNA damage in a PAR-dependent manner 7 – 12 .…”

The contribution of PARP1, PARP2 and poly(ADP-ribosyl)ation to base excision repair in the nucleosomal context

“…On the other hand, the interactions of PARP2 with the BER proteins have not been estimated quantitatively. Furthermore, the data on the affinity and activation of PARP1 and PARP2 towards damaged DNA indicate that PARP1 prefers earlier BER-intermediates, whereas PARP2 mostly acts on later ones (30, 31, 87). In addition, this study as well as literature data indicate functional dependence of various repair enzymes’ activities on PARylation and PAR synthesis (reviewed in 26).…”