Structural Characterization of Human Meiosis-Specific Protein-Protein Interfaces

Abstract

Meiosis produces haploid gametes essential for the process of sexual reproduction. This is achieved by cells undergoing a single round of DNA replication followed by two successive rounds of cell division: meiosis I and meiosis II. In meiosis I, replicated homologous chromosomes are paired, and subsequently separated into individual cells; in meiosis II the sister chromatids are separated, generating cells with half the usual chromosome content. As well as producing haploid gametes, the other critical function of meiosis is to generate genetic diversity. For this, during meiosis I, homologs must first pair correctly with their respective partners. Then, programmed double strand breaks are made and subsequently repaired using the other homolog as a template, leading to an exchange of genetic information. One hallmark of meiotic prophase I is the pairing of homologous chromosomes. This is promoted by chromosomal movement via the telomeres, the ends of linear chromosomes, which are tethered to the inner nuclear membrane. Telomeres are nucleoprotein complexes at the ends of chromosomes which have essential roles in a cell: to recruit telomerase to replicate the ends of chromosomes and to protect natural chromosome ends from evoking a DNA damage response. During meiosis, telomeres need to tether to the nuclear membrane so that cellular motors outside the nucleus can exert their force across the membrane and allow dynamic chromosome movements. I mapped a major interaction underlying telomere-inner nuclear membrane tethering by solving the crystal structure of the double stranded telomeric DNA binding protein TRF1 bound to meiosis specific protein TERB1. I demonstrated that TERB1 uses a TRF1-binding motif that resembles that of constitutive telomeric protein TIN2 through the X-ray crystallographic structure of the TRF1-TERB1 interface, solved at 2.1 Å resolution. This structure was validated using site-directed mutagenesis and biochemical assays as well as studies in mice, where mutations in the TERB1-TRF1 interface prevented telomere localization to the inner nuclear membrane and pairing of homologs. A key feature of meiosis is the chromosomal crossover events that occur which directly contribute to generating genetic diversity. Double stranded breaks are introduced into paired and synapsed chromosome homologs by the endonuclease spo11. Subsequently, these breaks are repaired using the other homolog as a template through homologous recombination (HR). A newly identified meiosis-specific protein named MEILB2 is essential to recruit HR protein BRCA2 to a meiotic DNA break. Subsequently, recombinases RAD51 and DMC1 are recruited to repair the break and complete HR. To better understand how MEILB2 and BRCA2 interact, I solved the 2.56Å X-ray crystallographic structure of MEILB2 in complex with the MEILB2 binding domain (MBD) of BRCA2. This structure revealed how two BRCA2 molecules bind two MEILB2 homodimers, a stoichiometry that agrees with the 4:2 MEILB2:BRCA2 complex that I observe in solution. The MEILB2 N terminus, which lies upstream of its armadillo repeats, mediates its homodimerization via a hydrophobic interface and a disulfide bond formed between conserved cysteine residues. Conserved salt bridges mediate the binding between BRCA2 and a single MEILB2 molecule from each homodimer. This structure provides the framework for testing the role of the MEILB2 homodimer and MEILB2-BRCA2 complex in meiotic HR. Together, my research has helped define key interfaces involving two novel meiosis-specific proteins that contribute to two critical goals in meiosis, namely, telomere-inner nuclear membrane tethering and meiotic HR.