DNA Repair 3 (2004) 301–312 Identification of specific amino acid residues in the E. coli  processivity clamp involved in interactions with DNA polymerase III, UmuD and UmuD ′ Jill M. Duzen a , Graham C. Walker b , Mark D. Sutton a,∗ a Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, 3435 Main Street, 140 Farber Hall, Buffalo, NY 14214, USA b Biology Department, 68-633 Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA Received 17 September 2003; received in revised form 18 November 2003; accepted 20 November 2003 Abstract Variants of a pentapeptide sequence (QL[S/F]LF), referred to as the eubacterial clamp-binding motif, appear to be required for certain proteins to bind specifically to the Escherichia coli  sliding clamp, apparently by making contact with a hydrophobic pocket located at the base of the C-terminal tail of each  protomer. Although both UmuC (DNA pol V) and the  catalytic subunit of DNA polymerase III (pol III) each bear a reasonable match to this motif, which appears to be required for their respective interactions with the clamp, neither UmuD not UmuD ′ do. As part of an ongoing effort to understand how interactions involving the different E. coli umuDC gene products and components of DNA polymerase III help to coordinate DNA replication with a DNA damage checkpoint control and translesion DNA synthesis (TLS) following DNA damage, we characterized the surfaces on  important for its interactions with the two forms of the umuD gene product. We also characterized the surface of  important for its interaction with the  catalytic subunit of pol III. Our results indicate that although UmuD, UmuD ′ and  share some common contacts with , each also makes unique contacts with the clamp. These findings suggest that differential interactions of UmuD and UmuD ′ with  impose a DNA damage-responsive conditionality on how  interacts with the translesion DNA polymerase UmuC. This is formally analogous to how post-translational modification of the eukaryotic PCNA clamp influences mutagenesis. We discuss the implications of our findings in terms of how E. coli might coordinate the actions of the umuDC gene products with those of pol III, as well as for how organisms in general might manage the actions of their multiple DNA polymerases. © 2003 Elsevier B.V. All rights reserved. Keywords: DNA replication; Translesion DNA synthesis; DNA polymerase management; umuDC;  sliding clamp; dnaN 1. Introduction The number of biochemically documented DNA poly- merases (pols) in both prokaryotic and eukaryotic organ- isms has increased dramatically in last few years, such that the gram negative bacterium Escherichia coli is currently known to possess 5 distinct DNA pols (reviewed in [1–3]), while humans are now known to possess at least 16 (re- viewed in [1–3]). The fact that each of these different pols possesses unique biochemical properties suggests that each may perform one or more specialized biological functions. However, very little is known about how a cell controls the ∗ Corresponding author. Tel.: +1-716-829-3581; fax: +1-716-829-2661. E-mail address: mdsutton@buffalo.edu (M.D. Sutton). actions of its different pols so that the appropriate enzyme gains access to the replication fork at the correct time. To some extent, it is possible to regulate which pol gains ac- cess to the replication fork by regulating the activity, the expression level, and/or the sub-cellular localization of the different pols within the cell. However, numerous studies performed using E. coli suggest that such controls are in- sufficient on their own, and that in addition to these levels of regulation, cells must also manage the actions of its dif- ferent pols via a higher order process that we have termed replication fork management [3–7]. Following replication blocking DNA damage in E. coli, RecA protein, which is the major bacterial recombinase, binds to single stranded (ss) DNA that accumulates, form- ing RecA-nucleoprotein filaments [8,9]. In addition to their role in homologous recombination [10,11], these filaments 1568-7864/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2003.11.008