Open sesame: activating dormant replication origins in the mouse immunoglobulin heavy chain (Igh) locus

Abstract

Chromosomal DNA replication in mammals initiates from replication origins whose activity differs in accordance with cell type and differentiation state. In addition to origins that are active in unperturbed conditions, chromosomes also contain dormant origins that can become functional in response to certain genotoxic stress conditions. Improper regulation of origin usage can cause genomic instability leading to tumorigenesis. We review findings from recent single-molecule DNA fiber studies examining replication of the mouse immunoglobulin heavy chain (Igh) locus, in which origin activity over a 400kb region is subject to dramatic developmental regulation. Possible models are discussed to explain such differential origin usage, particularly during replication stress conditions that can activate dormant origins.

Figure 1. SMARD analysis of the mouse Igh locus

A. (Top) Map of the 180 kb SwaI segment containing the constant region genes of the mouse Igh locus. (Bottom) Replication forks progress predominantly in one direction through the constant region of the Igh locus in mouse ES cells. Cells were sequentially labeled with IdU and CldU to reveal DNA replication tracts. Chromosomal DNA was subsequently isolated and analyzed by SMARD (e.g., see [22]). Images of single DNA molecules show regions of IdU (red tracts) and CldU (green tracts) incorporation. The blue regions show the position of hybridization probes utilized to identify and align the SwaI DNA segments. The DNA molecules have been arranged such that positions of the red-to-green transitions (yellow arrowheads) occur from right to left. These transitions correspond to the location of the DNA replication fork at the completion of the IdU labeling period. Initiation occurs downstream from these DNA molecules. B. In preB and proB cells, DNA replication initiates in the SwaI segment. These segments have internal origins indicated by the red patches surrounded by green. C. Insertion of a 10.7 kb initiation zone sequence into the SwaI element causes origin firing distal to the element. (Top) One SwaI molecule with DNA replication initiation sites was detected after the 10.7 kb sequence was inserted (green arrow). The merged image is shown above, with the individual channel images shown below. The earliest detected origin is indicated with a double white arrow. Two additional initiation events are also seen in the image. (Bottom) Another SwaI segment molecule with a replication initiation site observed outside of the 10.7 kb insertion.

Figure 1. SMARD analysis of the mouse Igh locus

A. (Top) Map of the 180 kb SwaI segment containing the constant region genes of the mouse Igh locus. (Bottom) Replication forks progress predominantly in one direction through the constant region of the Igh locus in mouse ES cells. Cells were sequentially labeled with IdU and CldU to reveal DNA replication tracts. Chromosomal DNA was subsequently isolated and analyzed by SMARD (e.g., see [22]). Images of single DNA molecules show regions of IdU (red tracts) and CldU (green tracts) incorporation. The blue regions show the position of hybridization probes utilized to identify and align the SwaI DNA segments. The DNA molecules have been arranged such that positions of the red-to-green transitions (yellow arrowheads) occur from right to left. These transitions correspond to the location of the DNA replication fork at the completion of the IdU labeling period. Initiation occurs downstream from these DNA molecules. B. In preB and proB cells, DNA replication initiates in the SwaI segment. These segments have internal origins indicated by the red patches surrounded by green. C. Insertion of a 10.7 kb initiation zone sequence into the SwaI element causes origin firing distal to the element. (Top) One SwaI molecule with DNA replication initiation sites was detected after the 10.7 kb sequence was inserted (green arrow). The merged image is shown above, with the individual channel images shown below. The earliest detected origin is indicated with a double white arrow. Two additional initiation events are also seen in the image. (Bottom) Another SwaI segment molecule with a replication initiation site observed outside of the 10.7 kb insertion.

Figure 1. SMARD analysis of the mouse Igh locus

A. (Top) Map of the 180 kb SwaI segment containing the constant region genes of the mouse Igh locus. (Bottom) Replication forks progress predominantly in one direction through the constant region of the Igh locus in mouse ES cells. Cells were sequentially labeled with IdU and CldU to reveal DNA replication tracts. Chromosomal DNA was subsequently isolated and analyzed by SMARD (e.g., see [22]). Images of single DNA molecules show regions of IdU (red tracts) and CldU (green tracts) incorporation. The blue regions show the position of hybridization probes utilized to identify and align the SwaI DNA segments. The DNA molecules have been arranged such that positions of the red-to-green transitions (yellow arrowheads) occur from right to left. These transitions correspond to the location of the DNA replication fork at the completion of the IdU labeling period. Initiation occurs downstream from these DNA molecules. B. In preB and proB cells, DNA replication initiates in the SwaI segment. These segments have internal origins indicated by the red patches surrounded by green. C. Insertion of a 10.7 kb initiation zone sequence into the SwaI element causes origin firing distal to the element. (Top) One SwaI molecule with DNA replication initiation sites was detected after the 10.7 kb sequence was inserted (green arrow). The merged image is shown above, with the individual channel images shown below. The earliest detected origin is indicated with a double white arrow. Two additional initiation events are also seen in the image. (Bottom) Another SwaI segment molecule with a replication initiation site observed outside of the 10.7 kb insertion.

Figure 2. Schematic of pre-RC complex formation and origin firing in mammals

In mammalian cells during the G1 phase of the cell cycle, the ORC hexameric complex serves as an interactive platform for the sequential recruitment of licensing factors including Cdc6, Cdt1 and Mcm9. This leads to eventual loading of multiple copies of the MCM2-7 complex onto chromatin, generating the replication competent pre-RC. Activation of cyclin-dependent kinases (CDK) after passing the restriction point facilitates the association of additional factors with the pre-RC to form the pre-initiation complex (not shown). CDK and Dbf4-dependent kinase (DDK) activities eventually lead to release of the replicative helicase formed by MCM2-7, Cdc45, and GINS, and generation of two replication forks that migrate bidirectionally outward from the origin. To reduce complexity in this schematic, we do not show most of the essential replication factors, including DNA polymerases that would associate with the new replication forks.

Figure 3. Models for differential origin activity

A. The first class of model postulates that a major determinant of origin activity is the occupancy of ORC binding. In the upper example, a weakly bound DNA element results in reduced ORC occupancy, as indicated by a transparent ORC (light blue oval). This reduced association would afford a correspondingly poorer licensing (less MCM loading) and little or no origin activity, leading to the DNA being replicated passively from a downstream origin (on the right). In the case of the 10.7 kb initiation region insertion into the murine Igh SwaI segment (second line), ORC occupancy could be increased by the ectopic DNA insert (green rectangle). In this example, the insert binds a factor (brown) that increases ORC binding (darker blue oval) and thereby causes weak but detectable origin activity (shown by a small replication bubble). Origin activity could occur outside of the insertion, because ORC binding may be increased in the general vicinity of the insert. Note that because of DNA looping, the bound ORC may have the potential to associate with MCM complexes bound to distal sequences, causing dormant origin activation at a distance (third line). The bottom example shows a DNA region that supports high occupancy of the ORC complex (dark blue oval) would also support efficient origin licensing (i.e., better MCM2-7 loading; red rings) and hence increased origin activity from that region (indicated by a large replication bubble). B. The second class of model proposes that the activity of ORC in loading the MCM complex, rather than the overall level of ORC binding, is the key feature that modulates origin activity. The top example indicates a DNA element that is unable to efficiently stimulate ORC activity, demonstrated by poorer loading of the MCM complex, and no origin activity. As in model A, the DNA would be replicated passively by a fork initiating from a downstream origin. Introduction of the ectopic element (middle example) provides a mechanism to stimulate ORC activity, leading to loading of additional MCM complexes and weak origin activity (a single small replication bubble). The bottom example depicts a DNA element that yields a highly-active ORC complex, indicated by the loading of multiple MCM2-7 complexes and efficient origin activity (signified by multiple replication bubbles along the DNA).