John J. Turchi

Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks

Restarting stalled replication forks partly depends on the break‐induced recombination pathway, in which a DNA double‐stranded break (DSB) is created on the stalled replication fork to initiate the downstream recombination cascades. Single‐stranded DNA gaps accumulating on stalled replication forks are potential targets for endonucleases to generate DSBs. However, it is unclear how this process is executed and which nucleases are involved in eukaryotic cells. Here, we identify a novel gap endonuclease (GEN) activity of human flap endonuclease 1 (FEN‐1), critical in resolving stalled replication fork. In response to replication arrest, FEN‐1 interacts specifically with Werner syndrome protein for efficient fork cleavage. Replication protein A facilitates FEN‐1 interaction with DNA bubble structures. Human FEN‐1, but not the GEN‐deficient mutant, E178A, was shown to rescue the defect in resistance to UV and camptothecin in a yeast FEN‐1 null mutant.

Replication Protein A (RPA) Binding to Duplex Cisplatin-damaged DNA Is Mediated through the Generation of Single-stranded DNA

Replication protein A (RPA) is a heterotrimeric protein composed of 70-, 34-, and 14-kDa subunits that has been shown to be required for DNA replication, repair, and homologous recombination. We have previously shown preferential binding of recombinant human RPA (rhRPA) to duplex cisplatin-damaged DNA compared with the control undamaged DNA (Patrick, S. M., and Turchi, J. J. (1998) Biochemistry 37, 8808–8815). Here we assess the binding of rhRPA to DNA containing site-specific cisplatin-DNA adducts. rhRPA is shown to bind 1.5–2-fold better to a duplex 30-base pair substrate containing a single 1,3d(GpXpG) compared with a 1,2d(GpG) cisplatin-DNA intrastrand adduct, consistent with the difference in thermal stability of DNA containing each adduct. Consistent with these data, a 21-base pair DNA substrate containing a centrally located single interstrand cisplatin cross-link resulted in less binding than to the undamaged control DNA. A series of experiments measuring rhRPA binding and concurrent DNA denaturation revealed that rhRPA binds duplex cisplatin-damaged DNA via the generation of single-stranded DNA. Single-strand DNA binding experiments show that rhRPA binds 3–4-fold better to an undamaged 24-base DNA compared with the same substrate containing a single 1,2d(GpG) cisplatin-DNA adduct. These data are consistent with a low affinity interaction of rhRPA with duplex-damaged DNA followed by the generation of single-stranded DNA and then high affinity binding to the undamaged DNA strand.

Cisplatin sensitizes cancer cells to ionizing radiation via inhibition of nonhomologous end joining

The combination of cisplatin and ionizing radiation (IR) treatment represents a common modality for treating a variety of cancers. These two agents provide considerable synergy during treatment, although the mechanism of this synergy remains largely undefined. We have investigated the mechanism of cisplatin sensitization to IR using a combination of in vitro and in vivo experiments. A clear synergistic interaction between cisplatin and IR is observed in cells proficient in nonhomologous end joining (NHEJ) catalyzed repair of DNA double-strand breaks (DSB). In contrast, no interaction between cisplatin and IR is observed in NHEJ-deficient cells. Reconstituted in vitro NHEJ assays revealed that a site-specific cisplatin-DNA lesion near the terminus results in complete abrogation of NHEJ catalyzed repair of the DSB. These data show that the cisplatin-IR synergistic interaction requires the DNA-dependent protein kinase–dependent NHEJ pathway for joining of DNA DSBs, and the presence of a cisplatin lesion on the DNA blocks this pathway. In the absence of a functional NHEJ pathway, although the cells are hypersensitive to IR, there is no synergistic interaction with cisplatin.

Human Ku autoantigen binds cisplatin-damaged DNA but fails to stimulate human DNA-activated protein kinase.

We have identified a series of proteins based on an affinity for cisplatin-damaged DNA. One protein termed DRP-1 has been purified to homogeneity and was isolated as two distinct complexes. The first complex is a heterodimer of 83- and 68-kDa subunits, while the second complex is a heterotrimer of 350-, 83-, and 68-kDa subunits in a 1:1:1 ratio. The 83- and 68-kDa subunits in each complex are identical. The 83-kDa subunit of DRP-1 was identified as the p80 subunit of Ku autoantigen by N-terminal protein sequence analysis and reactivity with a monoclonal antibody directed against human Ku p80 subunit. The 68-kDa subunit of DRP-1 cross-reacted with monoclonal antisera raised against the Ku autoantigen p70 subunit. The 350-kDa subunit was identified as DNA-PKcs, the catalytic subunit of the human DNA-activated protein kinase, DNA-PK. DRP-1/Ku DNA binding was assessed in mobility shift assays and competition binding assays using cisplatin-damaged DNA. Results indicate that DNA binding was essentially unaffected by cisplatin-DNA adducts in the presence or absence of DNA-PKcs. DNA-PK activity was only stimulated with undamaged DNA, despite the ability of Ku to bind to cisplatin-damaged DNA. The lack of DNA-PK stimulation by cisplatin-damaged DNA correlated with the extent of cisplatin-DNA adduct formation. These results demonstrate that Ku can bind cisplatin-damaged DNA but fails to activate DNA-PK. These results are discussed with respect to the repair of cisplatin-DNA adducts and the role of DNA-PK in coordinating DNA repair processes.

DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair

Replication protein A (RPA) is a heterotrimeric protein consisting of 70-, 34-, and 14- kDa subunits that is required for many DNA metabolic processes including DNA replication and DNA repair. Using a purified hyperphosphorylated form of RPA protein prepared in vitro, we have addressed the effects of hyperphosphorylation on steady-state and pre-steady-state DNA binding activity, the ability to support DNA repair and replication reactions, and the effect on the interaction with partner proteins. Equilibrium DNA binding activity measured by fluorescence polarization reveals no difference in ssDNA binding to pyrimidine-rich DNA sequences. However, RPA hyperphosphorylation results in a decreased affinity for purine-rich ssDNA and duplex DNA substrates. Pre-steady-state kinetic analysis is consistent with the equilibrium DNA binding and demonstrates a contribution from both the k(on) and k(off) to achieve these differences. The hyperphosphorylated form of RPA retains damage-specific DNA binding, and, importantly, the affinity of hyperphosphorylated RPA for damaged duplex DNA is 3-fold greater than the affinity of unmodified RPA for undamaged duplex DNA. The ability of hyperphosphorylated RPA to support DNA repair showed minor differences in the ability to support nucleotide excision repair (NER). Interestingly, under reaction conditions in which RPA is maintained in a hyperphosphorylated form, we also observed inhibition of in vitro DNA replication. Analyses of protein-protein interactions bear out the effects of hyperphosphorylated RPA on DNA metabolic pathways. Specifically, phosphorylation of RPA disrupts the interaction with DNA polymerase alpha but has no significant effect on the interaction with XPA. These results demonstrate that the effects of DNA damage induced hyperphosphorylation of RPA on DNA replication and DNA repair are mediated through alterations in DNA binding activity and protein-protein interactions.

Differential activation of DNA-PK based on DNA strand orientation and sequence bias

DNA-PKcs and Ku are essential components of the complex that catalyzes non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Ku, a heterodimeric protein, binds to DNA ends and facilitates recruitment of the catalytic subunit, DNA-PKcs. We have investigated the effect of DNA strand orientation and sequence bias on the activation of DNA-PK. In addition, we assessed the effect of the position and strand orientation of cisplatin adducts on kinase activation. A series of duplex DNA substrates with site-specific cisplatin–DNA adducts placed in three different orientations on the duplex DNA were prepared. Terminal biotin modification and streptavidin (SA) blocking was employed to direct DNA-PK binding to the unblocked termini with a specific DNA strand orientation and cisplatin–DNA adduct position. DNA-PK kinase activity was measured and the results reveal that DNA strand orientation and sequence bias dramatically influence kinase activation, only a portion of which could be attributed to Ku-DNA binding activity. In addition, cisplatin–DNA adduct position resulted in differing degrees of inhibition depending on distance from the terminus as well as strand orientation. These results highlight the importance of how local variations in DNA structure, chemistry and sequence influence DNA-PK activation and potentially NHEJ.

Interactions of mammalian proteins with cisplatin-damaged DNA

We have undertaken the systematic isolation and characterization of mammalian proteins which display an affinity for cisplatin-damaged DNA. Fractionation of human cell extracts has led to the identification of two classes of proteins. The first includes proteins that bind duplex DNA in the absence of cisplatin damage and retain their affinity for DNA in the presence of cisplatin-DNA adducts. The DNA-dependent protein kinase (DNA-PK) falls into this class. The inhibition of DNA-PK phosphorylation activity by cisplatin-damaged DNA has led to the hypothesis that cisplatin sensitization of mammalian cells to ionizing radiation may be mediated by DNA-PK. The second class of proteins identified are those which display a high relative affinity for cisplatin-damaged DNA and a low affinity for undamaged duplex DNA. Proteins that fall into this class include high mobility group 1 protein (HMG-1), replication protein A (RPA) and xeroderma pigmentosum group A protein (XPA). Each protein has been isolated and purified in the lab. The interaction of each protein with cisplatin-damaged DNA has been assessed in electrophoretic mobility shift assays. A series of DNA binding experiments suggests that RPA binds duplex DNA via denaturation and subsequent preferential binding to the undamaged DNA strand of the partial duplex. DNA substrates prepared with photo-reactive base analogs on either the damaged or undamaged DNA strand have also been employed to investigate the mechanism and specific protein-DNA interactions that occur as each protein binds to cisplatin-damaged DNA. Results suggest both damage and strand specificity for RPA and XPA binding cisplatin-damaged DNA.

High-mobility group 1 protein inhibits helicase catalyzed displacement of cisplatin-damaged DNA

We have determined the effect of HMG-1 bound to cisplatin-damaged DNA on the activities of calf helicase E. DNase I protection analysis demonstrated HMG-1 bound a cisplatin-damaged 24 base oligonucleotide annealed to M13mp18. Exonuclease digestion experiments revealed that greater than 90% of the DNA substrates contained a single site specific cisplatin adduct and, maximally, 65% of the substrates were bound by HMG-1. Helicase E catalyzed displacement of the cisplatin-damaged DNA oligonucleotide was inhibited by HMG-1 in a concentration-dependent manner. Time course experiments revealed a decreased rate of displacement in reactions containing HMG-1. The maximum inhibition observed was 55% and taking into account that only 65% of the substrates had HMG-1 bound, approximately 85% inhibition was observed on platinated DNA substrates containing HMG-1. Inhibition of helicase activity was proportional to the amount of substrate bound by HMG-1 based on the displacement and exonuclease assays at varying HMG-1 concentrations. The ability of helicase E to displace an undamaged DNA oligonucleotide from a cisplatin-damaged DNA template was also inhibited by HMG-1. Interestingly, HMG-1 had no effect on the rate of DNA-dependent ATP hydrolysis catalyzed by helicase E on the same DNA substrate. The inhibition of helicase activity by HMG-1 binding cisplatin-damaged DNA further supports a role for HMG-1 inhibiting DNA repair which may contribute to cellular sensitivity to cisplatin.