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

Abstract

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.

The distribution of alternating AT sequences in eukaryotic genomes suggests a role in homologous chromosome recognition in meiosis

“…They bind to the minor groove of DNA through a cluster of hydrophobic residues [32]. They may cover a long region of DNA through the formation of oligomers [33,34]. All these properties are consistent with specific binding to alternating AT clusters, in particular if they are unstabilized by torsional stress or cruciform formation: HMGB proteins always bind to distorted DNA regions.…”

“…Just as an eventual possibility, it can be mentioned that the Nhp6 proteins in yeast meet all the required properties. They may wrap around DNA duplexes [33] and have a preference for alternating AT sequences [30, Fig.4s]. They are present in the cell in sufficient amount and have multiple roles in chromosome structure and function [35].…”

Mechanisms of DNA binding determined in optical tweezers experiments

“…62 HMGB proteins have been observed to aid the formation of DNA loop complexes, similarly to the role of HU. 63,64 To explain these results, a simple model allows random transient binding to DNA, inducing random kinks that serve to compact the overall DNA structure. 62,64 Alternatively, HMG proteins may bind (perhaps being even unable to dissociate), changing the flexibility, possibly by locally denaturing the DNA as seen in recent experiments on the E. coli.…”

Transient HMGB protein interactions with B-DNA duplexes and complexes

“…DNA compaction by enhanced apparent flexibility suggests one general activity of HMGB proteins in vivo: reducing the effective persistence length of bulk DNA [39]. Such an effect would be important for stabilizing highly strained or distorted DNA conformations such as are encountered in recombination [29]. We have recently shown that bacterial cells lacking the architectural DNA binding protein HU are significantly disabled in lac operon repression looping [40], and expression of ectopic HMGB proteins can partially substitute for HU [40].…”

“…Studies by Johnson and co-workers [29] have shown that eukaryotic HMGB proteins can functionally replace bacterial architectural proteins in reconstituted in vitro recombination systems that require enhanced DNA bending. This and subsequent work [30;31;32;33;34;35; 36], suggests a model to explain how HMGB proteins enhance the rate of ligase-catalyzed DNA cyclization (Fig.…”