DNA ligase from the hyperthermophilic crenarcheon Sulfolobus shibatae (Ssh ligase) was optimally active in the presence of ATP and partially active in the presence of dATP. The enzyme was adenylated by both ATP and dATP, and the adenylate moiety covalently linked to the active site of the ligase was transferable to nicked DNA. Electrophoretic gel mobility shift assays revealed that the enzyme bound a duplex DNA fragment with a nick and that without a nick with similar affinity, but displayed little affinity for single-stranded DNA. Sso ligase, a closely related homologue of Ssh ligase from Sulfolobus solfataricus, interacted with PCNA-1, one of the three PCNA homologues found in the organism, as detected by yeast two-hybrid assays. No interaction of the enzyme with the other two PCNA homologues (PCNA-like and PCNA-2) was detected. Ssh10b, a member of the highly conserved Sac10b protein family of Archaea, stimulated DNA ligation by Ssh ligase, whereas Ssh7, a major Sulfolobus chromatin protein showed no effect on the activity of the ligase.