Specificity of SAF-A and lamin B binding in vitro correlates with the satellite DNA bending state.
There is evidence that Matrix Attachment Region (MAR)-binding proteins also bind satellite DNA (satDNA). The aim of the current work was to determine whether the major nuclear matrix (NM) MAR-binding proteins are able to recognize satDNAs of different locations and what DNA structural features are important for the recognition. In nuclei and NM, a number of the same polypeptides were recognized on a southwestern blot when MAR of immunoglobulin kappa gene (Ig kappa MAR) and pericentromeric (periCEN) satDNA fragments were used. However, the binding decreased dramatically when human and mouse CEN satDNA were used for the probes. After an NM extract was subjected to ion exchange chromatography, the main DNA-binding proteins were identified as SAF-A (scaffold attachment factor A) and lamin B. It was not possible to test the binding of lamin B by gel mobility shift assay (GMSA), but SAF-A showed an ability to distinguish CEN and periCEN satDNA fragments in GMSA. While periCEN fragments have an abnormally slow mobility on electrophoresis, which is a hallmark of bent DNA, CEN satDNA fragments have a normal mobility. A computer analysis was done using the wedge model (Ulanovsky and Trifonov [1987] Nature 326:720-722), which describes how the curved state depends on particular nucleotide sequences. The curved states of the fragments predicted by the model are in good agreement with their ability to be recognized by NM proteins. Thus SAF-A and lamin B are able to recognize conserved structural features of satDNA in the same way that MAR-binding proteins recognize MARs in spite of a lack of a consensus sequence. CEN and periCEN satDNAs are distinguished by proteins in correlation with the helical curvature of these fragments.
Lobov
I B
IB
Department of Cell Cultures, Institute of Cytology RAS, St. Petersburg 194064, Russia.
Tsutsui
K
K
Mitchell
A R
AR
Podgornaya
O I
OI
D000818:Animals; D015139:Blotting, Southern; D015342:DNA Probes / Q000032:analysis; D004276:DNA, Satellite / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D034921:Lamin Type B; D034882:Lamins; D008099:Liver / Q000378:metabolism; D051379:Mice; D015530:Nuclear Matrix / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004276:DNA, Satellite; D004268:DNA-Binding Proteins; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D012261:Ribonucleoproteins