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.

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G 2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G 2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.The human adeno-associated virus type 2 (AAV2) can perturb cell cycle progression (51, 71) and mediate specific killing of p53-deficient cells (51). Cells with intact p53 activity were able to arrest with 4N DNA content, whereas cells without functional p53 were not able to sustain this arrest and died. This effect was shown to depend not on the viral capsid proteins or other virus-encoded proteins but on the presence of the viral DNA. The AAV2 particle contains a single-stranded DNA molecule of 4.7 kb flanked by identical inverted terminal repeats which form T-shaped hairpin structures (5). The inverted terminal repeats are thought to function as primers for viral DNA replication. The hairpin structures of AAV2 DNA together with its single strandedness were hypothesized (51) to induce DNA damage signaling after AAV2 infection. In the work presented here, we set out to test this hypothesis, to identify proteins that recognize AAV DNA, and to elucidate how these proteins then activate the pathway that leads to G 2 arrest.An appropriate cellular response to DNA damage is crucial for maintenance of normal cell fate. Ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia-and Rad3-related (ATR) proteins are the two major signaling kinases that respond to DNA damage in cells.

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor ␣1 (GFR␣1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF 2 ⅐GFR␣1 2 complex structure at 2.35 Å resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF 2 ⅐GFR␣1 2 complex and the previously published artemin 2 ⅐GFR␣3 2 complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFR␣ interface between the two structures, in particular ones that buttress the key conserved Arg GFR␣ -Glu ligand -Arg GFR␣ interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFR␣s, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand 2 -co-receptor 2 -RET 2 heterohexamer complexes. Consistent with this, we showed that GDNF 2 ⅐GFR␣1 2 and artemin 2 ⅐GFR␣3 2 signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFR␣1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg 190 -Lys 202 region in GFR␣1 domain 2. This may explain how GDNF⅐GFR␣1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET. GDNF,3 originally characterized as a growth factor promoting the survival of midbrain dopaminergic neurons (1), regulates the differentiation and development of many peripheral neurons (2) and is neuroprotective (3). GDNF is also a morphogenic factor in kidney and spermatogonia development (reviewed by Airaksinen and Saarma (2)). Some clinical trials have indicated that perfusing GDNF into the putamen may be therapeutically beneficial in Parkinson disease (4). These neuroprotective and therapeutic roles have generated wide interest in the study of the GDNF signaling system.There are three other GDNF family ligands (GFLs), neurturin (NRTN (5)), artemin (ARTN (6)), and persephin (PSPN (7)), and knock-out mice experiments have made it clear that the order of biological importance is GDNF Ͼ Ͼ NRTN Ͼ ARTN Ͼ PSPN (2). They all signal primarily through the receptor tyrosine kinase RET (8). The extracellular region of RET has four cadherin-like domains and a cysteine-rich domain. Mutations in RET can cause both gain-of-function and loss-of-function diseases. In the former category are hereditary medullary thyroid carcinoma and multiple endocrine neoplasias of types 2A and 2B (9, 10), whereas Hirschsprung disease is an example of the latter (10).GFLs are distant relatives of transforming growth factor ␤ (2). Each GFL has its own co-receptor ␣: GDNF requires GFR␣1; N...

a b s t r a c tProtein splicing catalyzed by inteins has enabled various biotechnological applications such as protein ligation. Successful applications of inteins are often limited by splicing efficiency. Here, we report the comparison of protein splicing between 20 different inteins from various organisms in identical contexts to identify robust inteins with foreign exteins. We found that RadA intein from Pyrococcus horikoshii and an engineered DnaB intein from Nostoc punctiforme demonstrated an equally efficient splicing activity to the previously reported highly efficient DnaE intein from Nostoc punctiforme. The newly identified inteins with efficient cis-splicing activity can be good starting points for the further development of new protein engineering tools.