Lakhasly

7 have more plasticity in its origin selection142.Data from these newer mapping approaches show that replication origins can be kilobase- sized zones in metazoans and were reported to fire stochastically with no sequence specificity82,109,158.Consistently, metazoan ORC was shown to bind DNA with no apparent sequence specificity in vitro159-161.Despite the lack of sequence specificity, G-rich sequences which have the potential to form G-quadruplex secondary structure have been found to be associated with sites of efficientIn metazoan species, it has been more difficult to locate replication origins and independent studies with different methods do not show high concordance of origin features and locations90,151, likely due to the large genome sizes, the existence of large replication initiation zones, analysis of different cell types, movement of Mcm2-7 double hexamers and technical limitations.Interestingly, bioinformatic analysis of some other fungi, such as Neurospora crassa and Aspergillus nidulans, suggest that AT-hooks at their Orc4 N-terminus might be used to specify replication origin location at AT-rich sequences in their genomes (N. Zali and B. Stillman, unpublished).One possibility is that ORC from S. cerevisiae has lysine-rich sequence in an intrinsically disordered region (IDR) of the Orc1 subunit that binds to a minor grove in the origin DNA and this amino acid sequence is conserved in other eukaryotes and has been shown in ORC from human cells to bind DNA, possibly a short sequence the ORC1 IDR139,143.Following the release of the first draft of the human genome, techniques such as quantitative mapping of RNA-capped short nascent strands (SNS) and 2-dimensional gel electrophoresis were employed to map replication origins84,153-157.They are organized into spatially and temporally regulated replication timing domains6.

7
have more plasticity in its origin selection142. Strikingly, the Orc4 α-helix and the Orc2
loop are not found in ORC from most eukaryotes, including most fungi, plants and animals, raising the issue of how ORC from these species interacts with DNA and determines the location of origins of DNA replication. One possibility is that ORC from S. cerevisiae has lysine-rich sequence in an intrinsically disordered region (IDR) of the Orc1 subunit that binds to a minor grove in the origin DNA and this amino acid sequence is conserved in other eukaryotes and has been shown in ORC from human cells to bind DNA, possibly a short sequence the ORC1 IDR139,143.
In some fission yeasts, such as Schizosaccharomyces pombe (S. pombe), S. octosporus,
and S. japonicus, replication origins lack consensus sequences144,145 but are found to be AT-rich, which are called AT islands and can be as short as ~100bp in length to the size
of >500bp146,147. All these Schizosaccharomyces species contain an AT-hook domain at the N-terminus of the Orc4 protein that preferentially binds to AT-rich DNA sequences and is necessary for origin recognition148,149 (Table 2). There are nine AT-hook subdomains in S. pombe, while S. octosporus has four and S. japonicus has five146, and each subdomain is capable of binding 6–8 nucleotides of DNA150. Interestingly, bioinformatic analysis of some other fungi, such as Neurospora crassa and Aspergillus nidulans, suggest that AT-hooks
at their Orc4 N-terminus might be used to specify replication origin location at AT-rich sequences in their genomes (N. Zali and B. Stillman, unpublished). But again, like the DNA sequence specific origins that bind the Orc4 α-helix, the A-T-hook mechanism of origin specification is restricted to a small fraction of eukaryotic species, raising the question of how the location of origins of DNA replication are specified in most eukaryotes.
In metazoan species, it has been more difficult to locate replication origins and independent studies with different methods do not show high concordance of origin features and locations90,151, likely due to the large genome sizes, the existence of large replication initiation zones, analysis of different cell types, movement of Mcm2-7 double hexamers and technical limitations. In the late 1960s, visualization of replication tracts in mammalian cells revealed that replication initiates from multiple sites that can be simultaneously active at adjacent sites in the chromosomes152. Following the release of the first draft of the human genome, techniques such as quantitative mapping of RNA-capped short nascent strands (SNS) and 2-dimensional gel electrophoresis were employed to map replication origins84,153–157. In the past few years, many methods have been developed to map replication origins in metazoan genomes (Table 3).
Data from these newer mapping approaches show that replication origins can be kilobase- sized zones in metazoans and were reported to fire stochastically with no sequence specificity82,109,158. They are organized into spatially and temporally regulated replication timing domains6. Consistently, metazoan ORC was shown to bind DNA with no apparent sequence specificity in vitro159–161. Nevertheless, a recent study of replication origins
in mouse embryonic stem cells using a CRISPR-mediated deletion and inversion of
TAD boundaries suggest that cis-acting DNA elements are required for DNA replication initiation162. How these cis-acting sequences contribute to DNA replication is not known. Despite the lack of sequence specificity, G-rich sequences which have the potential to form G-quadruplex secondary structure have been found to be associated with sites of efficient