biostudies-literatureYalagapati SPGerman Research FoundationWIPANO Wissens und Technologietransfer durch Patente und Nomengkae1242https://www.ebi.ac.uk/biostudies/studies/S-EPMC11879092biostudies-literatureUnknown53(4)In eukaryotes, accurate chromosome segregation during cell division relies on the centromeric histone H3 variant, CENH3. Our previous work identified KINETOCHORE NULL2 (αKNL2) as a plant CENH3 assembly factor, which contains a centromere-targeting motif, CENPC-k, analogous to the CENPC motif found in CENP-C. We also demonstrated that αKNL2 can bind DNA in vitro in a sequence-independent manner, without the involvement of its CENPC-k motif. In this study, we show that the CENPC-k and CENPC motifs alone are insufficient for centromere targeting in Nicotiana benthamiana and Arabidopsis thaliana. In silico analysis identified adjacent DNA-binding regions near the CENPC-k and CENPC motifs, suggesting their role in centromeric DNA interaction. We further demonstrated that protein fragments contaNucleic acids researchCentromeric localization of αKNL2 and CENP-C proteins in plants depends on their centromere-targeting domain and DNA-binding regions.PMC1187909203THWST001LE2299/5-1LE2299/3-1Ahmadli UBerr ASinha ATalbert PLermontova IYalagapati SPDabravolski SZuo SKalidass MRutten TYadala RfalseCentromeric localization of αKNL2 and CENP-C proteins in plants depends on their centromere-targeting domain and DNA-binding regions.In eukaryotes, accurate chromosome segregation during cell division relies on the centromeric histone H3 variant, CENH3. Our previous work identified KINETOCHORE NULL2 (αKNL2) as a plant CENH3 assembly factor, which contains a centromere-targeting motif, CENPC-k, analogous to the CENPC motif found in CENP-C. We also demonstrated that αKNL2 can bind DNA in vitro in a sequence-independent manner, without the involvement of its CENPC-k motif. In this study, we show that the CENPC-k and CENPC motifs alone are insufficient for centromere targeting in Nicotiana benthamiana and Arabidopsis thaliana. In silico analysis identified adjacent DNA-binding regions near the CENPC-k and CENPC motifs, suggesting their role in centromeric DNA interaction. We further demonstrated that protein fragments conta2025-01-01T00:00:00Z2025 Feb2026-05-26T17:02:55.896Z2025-04-19T21:15:55.018ZS-EPMC118790923971898710.1093/nar/gkae1242