Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono-and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H 2 O 2 . A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.

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High-speed flight vehicles (HSFVs) such as space launch vehicles and missiles undergo severe dynamic loads which are generated during the launch and in in-flight environments. A typical vehicle is composed of thin plate skin structures with high-performance electronic units sensitive to such vibratory loads. Such lightweight structures are then exposed to external dynamic loads which consist of random vibration, shock, and acoustic loads created under the operating environment. Three types of dynamic loads (acoustic loads, rocket motor self-induced excitation loads and aerodynamic fluctuating pressure loads) are considered as major components in this study. The estimation results are compared to the design specification (MIL-STD-810) to check the appropriateness. The objective of this paper is to study an estimation methodology which helps to establish design specification for the dynamic loads acting on both vehicle and electronic units at arbitrary locations inside the vehicle.