The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein...

The mammalian high mobility group proteins HMG1 and HMG2 are abundant, chromatin-associated proteins whose cellular function is not known. In this study we show that these proteins can substitute for the prokaryotic DNA-bending protein HU in promoting the assembly of the Hin invertasome, an intermediate structure in Hin-mediated site-specific DNA inversion. Formation of this complex requires the assembly of the Hin recombinase, the Fis protein, and three cis-acting DNA sites, necessitating the looping of intervening DNA segments. Invertasome assembly is strongly stimulated by HU or HMG proteins when one of these segments is shorter than 104 bp. By use of ligase-mediated circularization assays, we demonstrate that HMG1 and HMG2 can bend DNA extremely efficiently, forming circles as small as 66 bp, and even 59-bp circles at high HMG protein concentrations. In both invertasome assembly and circularization assays, substrates active in the presence of HMG1 contain one less helical turn of DNA compared with substrates active in the presence of HU protein. Analysis of different domains of HMG1 generated by partial proteolytic digestion indicate that DNA-binding domain B is sufficient for both bending and invertasome assembly. We suggest that an important biological function of HMG1 and HMG2 is to facilitate cooperative interactions between cis-acting proteins by promoting DNA flexibility. A general role for HMG1 and HMG2 in chromatin structure is also suggested by their ability to wrap DNA duplexes into highly compact forms.