Biology subjects

Yalagapati, S. P.

Publications and source records attributed to Yalagapati, S. P..

2 recordsLinked to original sources

bioRxiv2024

Structural Basis of betaKNL2 Centromeric Targeting Mechanism and Its Role in Plant-Specific Kinetochore Assembly

The kinetochore is an essential protein complex that ensures proper chromosome segregation during cell division. Kinetochore assembly is initiated by the incorporation of CENP-A/CENH3. This process depends on KNL2/M18BP1 and CENP-C proteins. In plants, two variants of KNL2, KNL2 and {beta}KNL2, are present. Both possess the conserved SANTA domain, while KNL2 additionally has the centromere-targeting CENPC-k motif. Despite lacking the CENPC-like motif, the plant-specific {beta}KNL2 localizes to centromeres and aids in CENP-A/CENH3 loading. We found that efficient centromeric targeting of {beta}KNL2 requires the SANTA domain and the C-terminal part, while nuclear targeting depends on a conserved C-terminal motif-III. Structural predictions and experimental validations reveal that {beta}KNL2 forms homodimers and interacts with centromeric DNA and KNL2. We confirm that centromeric targeting of {beta}KNL2 depends on KNL2 in a tissue-dependent manner. Our findings provide crucial insights into the unique mechanisms of plant-specific kinetochore assembly, highlighting {beta}KNL2s essential role in this process. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/605747v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@30b331org.highwire.dtl.DTLVardef@14494a3org.highwire.dtl.DTLVardef@17a6336org.highwire.dtl.DTLVardef@7ad8f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Centromeric localization of KNL2 and CENP-C proteins in plants depends on their centromere-targeting domain and DNA-binding regions

In eukaryotic organisms, proper chromosome segregation during cell division depends on the centromeric histone H3 (CENH3) variant. Our previous studies identified a plant CENH3 assembly factor, Kinetochore Null2 (KNL2), that possesses a centromere-targeting motif, CENPC-k, similar to the CENPC motif in CENP-C. Additionally, we have demonstrated that KNL2 can bind DNA in vitro, independent of its CENPC-k motif. Thus, the mechanism underlying the binding of KNL2 to centromeric DNA remains elusive. Our study shows that the CENPC-k and CENPC motifs alone are not sufficient to target the centromere in N. benthamiana and A. thaliana. In-silico analysis revealed flanking DNA-binding regions near the CENPC-k and CENPC motifs, suggesting their importance in interacting with centromeric DNA. Fusion of protein fragments containing these motifs to EYFP facilitated targeting to the centromere. Deletion of DNA-binding domains reduced the centromeric localization of KNL2-C, whereas fusion of CENPC-k to the H-NS protein from E. coli targeted it to centromeres. We conclude that targeting of KNL2 and CENP-C proteins to centromeres is dependent on the CENPC-k/CENPC motifs, and their sequence-independent DNA-binding promotes anchoring at the centromere. Understanding the targeting mechanisms of KNL2 and CENP-C may help to engineer kinetochore structure by targeting chromatin modifying proteins to centromeres.

plant biology↗