DNA replication begins with the assembly of pre-replication complexes (pre-RCs) at thousands of DNA replication origins during the G1 phase of the cell cycle. At the G1-S-phase transition, pre-RCs are converted into pre-initiation complexes, in which the replicative helicase is activated, leading to DNA unwinding and initiation of DNA synthesis. However, only a subset of origins are activated during any S phase. Recent insights into the mechanisms underlying this choice reveal how flexibility in origin usage and temporal activation are linked to chromosome structure and organization, cell growth and differentiation, and replication stress.

Phosphorylation of H2AX (␥H2AX) is an early sign of DNA damage induced by replication stalling. However, the role of H2AX in the repair of this type of DNA damage is still unclear. In this study, we used an inactivated adeno-associated virus (AAV) to induce a stalled replication fork signal and investigate the function of ␥H2AX. The cellular response to AAV provides a unique model to study ␥H2AX function, because the infection causes pannuclear H2AX phosphorylation without any signs of damage to the host genome. We found that pannuclear ␥H2AX formation is a result of ATR overactivation and diffusion but is independent of ATM. The inhibition of H2AX with RNA interference or the use of H2AX-deficient cells showed that ␥H2AX is dispensable for the formation and maintenance of DNA repair foci induced by stalled replication. However, in the absence of H2AX, the AAV-containing cells showed proteosome-dependent degradation of p21, followed by caspase-dependent mitotic catastrophe. In contrast, H2AX-proficient cells as well as H2AX-complemented H2AX؊/؊ cells reacted by increasing p21 levels and arresting the cell cycle. The results establish a new role for H2AX in the p53/p21 pathway and indicate that H2AX is required for p21-induced cell cycle arrest after replication stalling.

The presence of CpG motifs and their associated sequences in bacterial DNA causes an immunotoxic response following the delivery of these plasmid vectors into mammalian hosts. We describe a biotechnological approach to the elimination of this problem by the creation of a bacterial cre recombinase expression system, tightly controlled by the arabinose regulon. This permits the Cre-mediated and -directed excision of the entire bacterial vector sequences from plasmid constructs to create supercoiled gene expression minicircles for gene therapy. Minicircle yields using standard culture volumes are sufficient for most in vitro and in vivo applications whereas minicircle expression in vitro is significantly increased over standard plasmid transfection. By the simple expedient of removing the bacterial DNA complement, we significantly reduce the size and CpG content of these expression vectors, which should also reduce DNA-induced inflammatory responses in a dose-dependent manner. We further describe the generation of minicircle expression vectors for mammalian mitochondrial gene therapy, for which no other vector systems currently exist. The removal of bacterial vector sequences should permit appropriate transcription and correct transcriptional cleavage from the mitochondrial minicircle constructs in a mitochondrial environment and brings the realization of mitochondrial gene therapy a step closer.There is increasing evidence to suggest that plasmid DNA used for non-viral gene delivery can cause unacceptable inflammatory responses in eukaryotes (1-5). These immunotoxic responses are largely due to the presence of unmethylated CpG motifs and their associated stimulatory sequences on plasmids, following bacterial propagation of plasmid DNA. Simple methylation of DNA in vitro may be enough to reduce an inflammatory response but is likely to result in severely depressed gene expression (6). The removal of CpG islands by cloning out, or elimination of non-essential sequences is more successful in reducing inflammatory responses but is time-consuming and tedious (7).Because bacterial DNA contains on average four times more CpG islands than does mammalian DNA (8), a good solution is to entirely eliminate the bacterial control regions from gene delivery vectors during the process of plasmid production. Removal of bacterial sequences needs to be efficient, using the smallest possible excision site, while creating supercoiled DNA minicircles, consisting solely of gene expression elements under appropriate mammalian control regions.This can be achieved by the use of Cre recombinase, a bacteriophage P1-derived integrase (9 -11), catalyzing site-specific recombination between direct repeats of 34 base pairs (loxP sites).In the case of a supercoiled plasmid containing DNA flanked by two loxP sites in the same orientation, Cre recombination produces two DNA molecules that are topologically unlinked, circular, and mainly supercoiled (10), each containing a single 34-bp 1 loxP site. Efficient minicircle production requires the use of...

Adeno-associated virus type 2 (AAV2) provokes a DNA damage response that mimics a stalled replication fork. We have previously shown that this response is dependent on ataxia telangiectasia-mutated and Rad3-related kinase and involves recruitment of DNA repair proteins into foci associated with AAV2 DNA. Here, we investigated whether recombinant AAV2 (rAAV2) vectors are able to produce a similar response. Surprisingly, the results show that both single-stranded and double-stranded green fluorescent protein-expressing rAAV2 vectors are defective in producing such a response. We show that the DNA damage signaling initiated by AAV2 was not due to the virus-encoded Rep or viral capsid proteins. UV-inactivated AAV2 induced a response similar to that of untreated AAV2. This type of DNA damage response was not provoked by other DNA molecules, such as single-stranded bacteriophage M13 or plasmid DNAs. Rather, the results indicate that the ability of AAV2 to produce a DNA damage response can be attributed to the presence of cis-acting AAV2 DNA sequences, which are absent in rAAV2 vectors and could function as origins of replication creating stalled replication complexes. This hypothesis was tested by using a single-stranded rAAV2 vector containing the p5 AAV2 sequence that has previously been shown to enhance AAV2 replication. This vector was indeed able to trigger DNA damage signaling. These findings support the conclusion that efficient formation of AAV2 replication complexes is required for this AAV2-induced DNA damage response and provide an explanation for the poor response in rAAV2-infected cells.