The Inherent Asymmetry of DNA Replication

Abstract

Semiconservative DNA replication has provided an elegant solution to the fundamental problem of how life is able to proliferate in a way that allows cells, organisms, and populations to survive and replicate many times over. Somewhat lost, however, in our admiration for this mechanism is an appreciation for the asymmetries that occur in the process of DNA replication. As we discuss in this review, these asymmetries arise as a consequence of the structure of the DNA molecule and the enzymatic mechanism of DNA synthesis. Increasing evidence suggests that asymmetries in DNA replication are able to play a central role in the processes of adaptation and evolution by shaping the mutagenic landscape of cells. Additionally, in eukaryotes, recent work has demonstrated that the inherent asymmetries in DNA replication may play an important role in the process of chromatin replication. As chromatin plays an essential role in defining cell identity, asymmetries generated during the process of DNA replication may play critical roles in cell fate decisions related to patterning and development.

Keywords: DNA replication; asymmetric; chromatin; epigenetic inheritance; histones.

Figure 1. Overview of DNA replication

The fork fires as the MCM2-7 helicase proceeds out in a bidirectional fashion from the origin of replication (ORI), unwinding double-stranded DNA as it goes. ORC proteins and cdc6 dissociated from the origin, where they were initially bound to facilitate helicase loading. CDC45 and GINS travel with the MCM2-7 helicase to create the CMG replication complex. The leading strand is synthesized primarily through the actions of DNA polymerase ε. The lagging strand is discontinuously synthesized primarily through the actions of DNA polymerase α and δ. Pol α synthesizes the RNA primer needed to initiate DNA synthesis. The lagging strand is additionally processed by FEN1 and ligase to complete synthesis. The RFC complex is responsible for loading PCNA, which acts as a processivity factor for DNA pol ε and DNA pol δ. Cohesins link the two sister chromatids following passage of the replication fork.

Figure 2. Mutational Imbalance during DNA replication

Mutagenesis occurs asymmetrically on the two sisters during DNA replication. The lagging strand may have a higher rate of mutagenesis than the leading strand due to both incorporation of low fidelity DNA Polymerase α and the strands increased susceptibility to mutagenesis with head on transcription and DNA replication fork collisions.

Figure 3. Asymmetries in DNA replication underlie mating-type switching in S. pombe

(A) A P-type mating cell entering DNA replication with the mat1 (mating type) locus labeled with blue circle. (A′) The replication fork approaches the mat1 locus. (A″) As replication proceeds, one strand is replicated by continuous (leading strand) synthesis whereas the other strand is replicated by discontinuous (lagging-strand) synthesis, characterized by numerous RNA primers. (A‴) One of these primers fails to get properly removed during lagging strand processing, leaving a molecular lesion (two RNA nucleotides) necessary for mating-type switching to occur. (A‴′) Two sister chromatids now exist: one with a molecular lesion and one which is unmarked. (B–C) The two sister chromatids are segregated to distinct daughter cells during mitosis. (D) Two daughter cells now enter S-phase; one with the molecular lesion and one without. (D′) In the daughter cell with the molecular lesion, the replication fork approaches the mat1 locus. (D″) The replication fork stalls when it hits the lesion. (D‴) The fork is rescued in DNA repair mechanism resembling synthesis dependent strand annealing. The result of this process is that the strand where the collision occurred has new DNA copied into the mat1 locus, resulting in a switch in mating type from P ➔ M. (D‴′) Two sister chromatids are produced; one which has switched mating type, the other which now contains a molecular lesion. (E) Two daughter cells are produced: one which has switch mating type, the other of which has not.

Figure 4. Asymmetries in DNA replication effect chromatin maturation following fork passage

(A) The replisome approaches the bound transcription factor with Pol II transcribing in the same direction as the fork. The approaching helicase causes the transcription factor to dissociate. Due to the fact that the lagging strand is still being processed, the transcription factor will preferentially bind the leading strand. (B) Once bound, the transcription factor can initiate transcription, which helps structure and order the nucleosomes on the leading strand. (C) The leading strand now has ordered, phased nucleosomes whereas the lagging strand, due to lack of transcription, still has disordered nucleosomes.

Figure 5. Asymmetric chromatin maturation leads to epigenetic asymmetries on sister chromatids

(A) One the leading strand, the transcription factor (TF) binds and initiates transcription, which helps structure and order the nucleosomes on the leading strand. On the lagging strand, TF binding fails to occur in a timely manner, allowing nucleosomes to encroach upon binding site. (B) Transcription on the leading strand leads to deposition of marks associated with transcription such as H3 variant H3.3 and tri-methylation of the 4^th lysine on histone H3 (H3K4me3). The transcriptionally-inert lagging strand receives none of these chromatin modifications.

Figure 6. Cohesins maintain nucleosome free regions (NFRs) in the wake of the replication fork

(A) The replisome approaches a bound transcription factor, causing the transcription factor to dissociate. (B) Cohesin binds immediately after the passage of the replication fork and prevents histones from invading transcription factor binding sites, maintaining them as nucleosome free regions. (C) Transcription factors can now bind leading and lagging strands and initiate synthesis in the wake of replication fork passage.

Figure 7. Overview of replication-coupled nucleosome assembly

The MCM2-7 helicase sits at the foremost edge of the replication fork where its primary function is to unwind non-replicated DNA. Torsional strain ahead of the fork begins to break apart nucleosomes, which must then be recycled to newly synthesized DNA on the other side of the fork. The MCM2 protein can bind (H3–H4)₂ tetramers dissociated in the wake of the advancing fork to allow for their subsequent deposition on nascent DNA. Due to its ability to bind (H3–H4)₂ tetramers, CAF-1 may help coordinate the deposition of preexisting histones after fork passage. CAF-1 is recruited to the edge of the advancing fork by PCNA, which also serves as a processivity factor for the replicative polymerases ε and δ. CAF-1 then coordinates new H3–H4 deposition onto nascent DNA. The subsequent incorporation of two (H2A-H2B) dimers reforms the nucleosome structure and signifies the end of the process of replication coupled histone deposition.

Figure 8. Different models for histone inheritance

(A) Semi-conservative model: Old (H3–H4)₂ tetramers are split at the replication fork, allowing (H3–H4) dimers to be inherited equally on both the leading and the lagging strand. New (H3–H4) dimers pair with old (H3–H4) dimers to recreate the tetramer structure. (B) Dispersive model: Old (H3–H4)₂ tetramers remain together at the fork and are randomly segregated to leading or lagging strand in roughly equal numbers. New histones fill in gaps left by old histones to reconstitute nucleosome density. (C) Conservation model: Old (H3–H4)₂ tetramers remain un-split at the fork and are inherited as a tetramer. In this model, tetramers are biased in their inheritance such that either the leading strand (shown) or the lagging strand (not shown) inherits a majority of the old (H3–H4)₂ tetramers. New histones fill in gaps left by old histones to reconstitute nucleosome density.

Figure 9. Old histone distribution in the absence of new histone synthesis

(A) In the event that old histones have an equal likelihood of binding the leading or lagging strand, old histones should distribute themselves on the leading and lagging strands in roughly equal numbers. (B) If the likelihood of stable binding the leading strand is higher than on the lagging strand, then more old histones should be bound to the leading strand in a manner that is proportional to increased favorability of leading strand association. (C) If the lagging strand is incapable of binding old histones in the immediate aftermath of replication fork passage, then all old histones should be recycled onto the leading strand.

Figure 10. Hypothetical asymmetric nucleosome density in leading vs. lagging strand

As PCNA has been shown to play a central role in recruiting new histones for deposition on newly synthesized DNA, the increased density of PCNA on the lagging strand could serve to increase histone density on the lagging strand when compared to the leading strand. Increased histone density would chance the chromatin state of these regions, possibly as a way to bias the transcriptional output of the two sister chromatids.

Figure 11. Asymmetric vs. Symmetric histone distribution in Drosophila melanogaster germline stem cells (GSC) sister chromatids

(A) Old H3 are enriched in the sister chromatids inherited by the cell fated to remain a GSC whereas newly-synthesized H3 are enriched in the sister chromatids inherited by the daughter cell destined to differentiate. (B) Old and new H3.3 are present in equal quantities on all sister chromatids and are inherited equally by GSC and non-GSC daughter cells.

Figure 12. Asymmetric vs. symmetric epigenetic regions on metaphase sister chromatids

(A) Epigenetically asymmetric gene locus. Epigenetic information is asymmetrically partitioned between the two sister chromatids. Age of histone is shown as a representative epigenetic asymmetry. Other epigenetic modifications such as histone post-translational modifications, nucleosome density, and histone variant incorporation could also establish epigenetically distinct sister chromatid regions. (B) Epigenetically symmetric gene locus. Epigenetic modifications are equally distributed between sister chromatids.

Figure 13. Segregation of asymmetrically modified sister chromatids vs. symmetrically modified sister chromatids during asymmetric cell division

(A) Asymmetric cell division. Sister chromatids bearing asymmetric epigenetic information at key developmental loci are selectively recognized and segregated by asymmetric modifications in the centromeric region. These asymmetries in the centromere could allow the microtubules to bind and recognize these molecularly-distinct sisters in order to segregate them to the appropriate daughter cell. (B) Symmetric cell division. Sister chromatids containing identical epigenetic information have identical centromere structures, and are segregated randomly during the process of cell division.