DNA replication process in eukaryotes follows a highly reproducible temporal programme, with some genomic regions being replicated earlier and some later in the course of the S phase. Appropriate execution of this program is of key importance for proper replication and maintenance of genomic integrity. In the past years, Rif1, originally discovered as telomere associated protein, has emerged as one of the crucial regulators of replication timing program in both yeast and higher eukaryotes. Rif1 has been shown to act as a negative regulator of replication initiation at certain origins and has an effect on replication timing genome wide. In this work we have set out to better understand key functional parts of Rif1 domains and potentially further examine if these are involved in DNA binding, or instead serve as interaction motifs for the recruitment of as yet unidentified proteins. Additionally, we are implying innovative approaches in order to resolve yet unsolved question behind Rif1 detection at replication origins in Saccharomyces cerevisiae.