OBA/Ku86: DNA Binding Specificity and Involvement in Mammalian DNA Replication

Abstract

Ors-binding activity (OBA) was previously semipurified from HeLa cells through its ability to interact specifically with the 186-basepair (bp) minimal replication origin of ors8 and support ors8 replication in vitro. Here, through competition band-shift analyses, using as competitors various subfragments of the 186-bp minimal ori, we identified an internal region of 59 bp that competed for OBA binding as efficiently as the full 186-bp fragment. The 59-bp fragment has homology to a 36-bp sequence (A3/4) generated by comparing various mammalian replication origins, including the ors. A3/4 is, by itself, capable of competing most efficiently for OBA binding to the 186-bp fragment. Band-shift elution of the A3/4 -OBA complex, followed by Southwestern analysis using the A3/4 sequence as probe, revealed a major band of ϳ92 kDa involved in the DNA binding activity of OBA. Microsequencing analysis revealed that the 92-kDa polypeptide is identical to the 86-kDa subunit of human Ku antigen. The affinity-purified OBA fraction obtained using an A3/4 affinity column also contained the 70-kDa subunit of Ku and the DNA-dependent protein kinase catalytic subunit. In vitro DNA replication experiments in the presence of A3/4 oligonucleotide or anti-Ku70 and anti-Ku86 antibodies implicate Ku in mammalian DNA replication. INTRODUCTIONKu antigen (autoantigen) is a heterodimeric (p70/p86) DNA-binding protein recognized by autoantibodies from the sera of certain patients with systemic rheumatic diseases (Mimori et al., 1981;Reeves, 1985;Yaneva et al., 1985;Mimori and Hardin, 1986). It consists of two polypeptides of 86 and 70 kDa (Yaneva et al., 1985). Ku is identical to a DNA-dependent ATPase isolated from HeLa cells (Cao et al., 1994) that had been previously reported to cofractionate with a 21S multiprotein complex competent for DNA synthesis from HeLa cells (Vishwanatha and Baril, 1990). Furthermore, the interaction of Ku antigen with a human DNA region (B48) containing a replication origin was reported (Tóth et al., 1993), and a novel ATP-dependent DNA unwinding enzyme, DNA helicase II (HDH II), was identified as Ku (Tuteja et al., 1994). Recently, Ochem et al. (1997) reported that the Ku70 subunit is the one associated with the helicase activity in the Ku70/Ku86 heterodimer. Moreover, a role for Ku70 as a tumor suppressor for murine T cell lymphoma has been suggested, because Ku70 deficiency facilitates neoplastic growth (Li et al., 1998). Ku has been shown to be the DNA-binding subunit of the DNA-dependent protein kinase (DNA-PK) holoenzyme Suwa et al., 1994), a nuclear component that phosphorylates a number of DNA-binding, regulatory proteins, including transcription factors (Sp1, p53), RNA polymerase II, topoisomerases I and II, Ku antigen, and SV-40 large T antigen (Anderson, 1993, and references therein). Although Ku has been characterized as a DNA end-binding protein, it was recently shown that it is also a sequence-specific DNA-binding protein, binding to negative regulatory element 1 (NRE1) in the long terminal repeat of...

The DNA End-Binding Protein Ku Regulates Silencing at the InternalHMLandHMRLoci inSaccharomyces cerevisiae

“…Taken together with previous observations, our results suggest that Ku is endowed with two modes of binding to silent regions of DNA: it can bind telomeric regions directly via its DNA end-binding activity and it can bind HML and HMR as a result of protein-protein interactions. Previous observations have also implicated Ku in binding to internal chromosomal loci and suggest that Ku may play a role in the activation of transcription and possibly in initiation of replication (Barnes and Rio 1997;Ruiz et al 1999;Novac et al 2001;Walker et al 2001;Schild-Poulter et al 2003;Sibani et al 2005;Grote et al 2006;Shi et al 2007;Rampakakis et al 2008). The data presented here support and extend the data indicating that Ku binds to internal chromosomal loci and broaden our knowledge of the number of processes in which Ku plays a role at internal loci to include silencing.…”

Decreased origin usage and initiation of DNA replication in haploinsufficient HCT116 Ku80+/- cells

“…Purified as an origin binding activity (OBA) from HeLa cells (Ruiz et al, 1995;Ruiz et al, 1999) through its ability to bind to the minimal functional sequence of the monkey replication origin ors8 (Frappier and ZannisHadjopoulos, 1987;Landry and Zannis-Hadjopoulos, 1991;Pearson et al, 1991;Todd et al, 1995), OBA was identified by microsequencing as Ku80 (Ruiz et al, 1999). Since then, several studies implicated Ku in the initiation of DNA replication (Novac et al, 2001;Matheos et al, 2002) , and refs therein].…”

“…Since then, several studies implicated Ku in the initiation of DNA replication (Novac et al, 2001;Matheos et al, 2002) , and refs therein]. In human (HeLa) cells, Ku was shown to co-fractionate with the 21S multiprotein complex competent for DNA synthesis (Vishwanatha and Baril, 1990) and to bind replication origins in a sequence-specific manner (Toth et al, 1993;Ruiz et al, 1999; …”

Synergy Between DNA Replication and Repair Mechanisms

“…In contrast, their wild-type cells continued to synthesize DNA and were able to promptly repair the DNA breaks, suggesting a role of DNA-PK in immediately repairing DNA breaks following deceleration of DNA replication. Altogether these results suggest that, in addition to its role in repairing dsDNA breaks that occur during replication fork progression (Shimura et al, 2007), Ku is also involved in the prevention of DNA breaks caused by replication fork collapse by: i) binding onto DNA replication origins at G1 phase (Novac et al, 2001;Ruiz et al, 1999), recruiting the DNA replication machinery (Rampakakis et al, 2008;Rampakakis and Zannis-Hadjopoulos, 2009;Sibani et al, 2005b) and ensuring genomic duplication and maintenance (Toth et al, 1993) (progression into S phase without the appropriate number of activated replication origins would lead to an increase of the average replicon size, resulting in stalled replication forks and chromosomal instability (Ekholm-Reed et al, 2004;Tanaka and Diffley, 2002a)); and ii) maintaining the DNA polymerase processivity factor PCNA on chromatin following ionizing radiation (Park et al, 2004).…”

“…Ku is an origin binding protein, binding to several replication origins, among them the adenovirus type 2 origin (de Vries et al, 1989), the Herpes Simplex Virus Type 1 (HSV1) origin (Murata et al, 2004), the B48 human origin (Toth et al, 1993), the mammalian replication origin consensus sequence, A3/4 Ruiz et al, 1999), the Chinese hamster dihydrofolate reductase (DHFR) replication origin, ori, and the monkey replication origins ors8 and ors12 (Novac et al, 2001), as well as the human origins lamin B2, -globin, c-myc (Sibani et al, 2005a, b) and dnmt1 (DNA-methyltransferase) (Araujo et al, 1998). Ku was shown to associate in vivo with replication origins in a cell cycle dependent manner (Novac et al, 2001;Ruiz et al, 1999;Sibani et al, 2005a) and its differential binding to DNA is a determining factor in its involvement in DNA replication, exhibiting distinct origin DNA binding properties from its association with DNA ends or other internal DNA sequences (Schild-Poulter et al, 2003). The role of Ku in DNA replication is believed to be two-fold.…”