Investigations of the pathway of incorporation and function of lamin A in the nuclear lamina

Cell-free extracts of Xenopus eggs were used to investigate how differing l amin sub-types are incorporated into a lamina and how lamina composition in fluences DNA replication. Purified recombinant human lamin A (HlaminA) was inoculated into egg extracts, which support in vitro nuclear assembly. The route of incorporation of Hlamin A into the lamina was compared to Xenopus lamin B-3 (XlaminB(3)), the major endogenous lamin sub-type in egg extracts . While Xlamin B-3 was incorporated into the lamina directly, HlaminA first accumulated at nucleoplasmic foci, before entering the lamina. When Hlamin A was inoculated into extracts depleted of XlaminB(3) it entered nuclei eff iciently and was incorporated into nucleoplasmic foci. However, in the abse nce of XlaminB(3), HlaminA remained in the foci and did not enter the nucle ar envelope. When HlaminA entered the nuclear envelope, it did not influenc e DNA replication. Nuclei containing HlaminA initiated DNA replication on q ueue and in addition the spatial distribution of replication centres in the se nuclei was identical to controls. Taken together, these results suggest that the incorporation of A-type lamins into the nuclear envelope is depend ent upon the presence of B-type lamins. However, the presence of A-type lam ins alone is not sufficient to influence the spatial and temporal order of DNA replication. (C) 1999 Wiley-Liss, Inc.