GST-lamin fusion proteins act as dominant negative mutants in Xenopus egg extract and reveal the function of the lamina in DNA replication

Abstract

A cDNA encoding Xlamin B1 was cloned from a whole ovary mRNA by RT-PCR. GST-lamin fusion constructs were generated from this cDNA by first creating convenient restriction sites within the Xlamin B1 coding sequence, using PCR directed mutagenesis, and then sub-cloning relevant sequences into pGEX-4T-3. Two expression constructs were made, the first, termed delta 2+ lacked sequences encoding the amino-terminal 'head domain' of lamin B1 but included sequences encoding the nuclear localization signal sequence (NLS). The second expression construct, termed delta 2-, lacked sequences encoding the amino-terminal 'head domain' as well as sequences encoding the NLS. Purified fusion proteins expressed from these constructs, when added to egg extracts prior to sperm pronuclear assembly, formed hetero-oligomers with the endogenous lamin B3. The delta 2+ fusion protein prevented nuclear lamina assembly but not nuclear membrane assembly. The resulting nuclei were small (approximately 10 microns in diameter), did not assemble replication centers and failed to initiate DNA replication. When the delta 2- fusion protein was added to egg extracts prior to sperm pronuclear assembly, lamina assembly was delayed but not prevented. The resulting nuclei although small (approximately 12 microns), did form replication centers and initiated DNA replication. When added to egg extracts after sperm pronuclear assembly was completed delta 2+, but not delta 2-, entered the pre-formed nuclei causing lamina disassembly. However, the disassembly of the lamina by delta 2+ did not result in the disruption of replication centers and indeed these centres remained functional. These results are consistent with the hypothesis that lamina assembly precedes and is required for the formation of replication centers but does not support those centers directly.