Lecture 6 Part 1 Notes: Origins of Replication, Histone Inheritance, and Telomeres

Origins of Replication in Eukaryotes

DNA replication begins at origins of replication; in human cells, each chromosome has many origins to speed up replication by enabling multiple origins to be active concurrently. Not all origins fire at once; origins in euchromatin tend to replicate first, but many origins operate at the same time.
To start replication at origins, the DNA double helix must be melted open to allow access for replication machinery. Initiator proteins pry apart double-stranded DNA.
Initiation must be tightly regulated so that it occurs once per cell division and does not alter copy number.

In a eukaryotic origin of replication diagram, during G1 (gap phase 1) a protein complex is bound to the origin: the origin recognition complex, ORC, bound to DNA together with Cdc6.
Later in G1, Cdc6 is lost and the Mcm helicase is loaded onto the DNA. Together with ORC, this forms the pre-replicated complex (the licensed origin).
At the G1/S boundary (the restriction point), a change occurs: both ORC and Mcm helicase are phosphorylated. This activates the helicase, which launches and melts the DNA double helix.
The ORC is temporarily displaced, DNA polymerase and the rest of the DNA replication machinery are recruited, and DNA synthesis begins.
The phosphorylated ORC rebinds the DNA, but in this phosphorylated state it cannot initiate another round of replication at this origin.
Relicensing for another round of replication does not occur until cell division completes and daughter cells return to G1 and ORC becomes dephosphorylated.

In addition to copying DNA sequence, a second code exists: the histone code from histone tail modifications (acetylation, phosphorylation, methylation, etc.).
These modifications influence gene expression and are inherited by daughter cells, helping define cell identity and phenotype.

As the replication fork progresses, histones around which DNA is wrapped are displaced and partially disassembled.
The histone H3:H4 tetramers tend to stay associated with the DNA and are randomly inherited by each daughter cell.
The histone H2A:H2B dimers are displaced from the DNA and are scattered; they mix with newly synthesized histones.
In human cells, there are 20 copies of each histone gene, enabling rapid synthesis of histones when needed.

After replication, nucleosome assembly is aided by histone chaperones NAP1 and CAF1 and their interaction with the sliding clamp.
NAP1 handles loading of H2A:H2B dimers; CAF1 handles loading of H3:H4 tetramers, restoring chromatin on both daughter strands.

Eukaryotic chromosomes are linear, so they have ends. DNA polymerase requires an RNA primer to initiate synthesis, and the final RNA primers cannot be replaced with DNA due to the lack of a 3' hydroxyl end for the repair polymerase.
Without a solution, ends would progressively shorten with each replication.
Prokaryotes avoid this problem via circular genomes; eukaryotes solve it with telomeres and telomerase.

Telomerase is a DNA polymerase that carries its own RNA template (telomerase RNA) and uses it to extend telomeres via RNA-templated DNA synthesis, resembling reverse transcriptase.
Telomerase activity helps extend the 3' overhang at telomeres, preventing progressive shortening.

Telomeres consist of repetitive DNA sequences; in humans, the repeat is roughly GGG TTA, repeated about 10^3 times (approximately 1000 repeats).
The telomere ends with a 3' overhang on the parent strand (the template strand for replication).
Telomerase extends this 3' overhang, after which DNA polymerase alpha (which comes with its own primase) completes the lagging strand; this process leaves a residual 3' overhang but preserves telomere length.
The extended 3' overhang can fold back to form a loop (the T loop) through strand invasion, helping to protect the chromosome end.

The T loop and the telomeric region are covered by a protective protein cap called shelterin.
Shelterin shields telomeres from DNA damage repair mechanisms, preventing accidental chromosome fusions and maintaining end integrity.

Telomeres shorten gradually in human somatic cells, losing about 100\text{ to }200 base pairs per division.
- This shortening can lead to replicative senescence when telomeres become too short to function properly, acting as a safeguard against unlimited cell proliferation and cancer.
Stem cells in tissues that renew themselves (e.g., bone marrow, gut) retain full telomerase activity and can undergo more divisions.

Links to chromosome structure: origin licensing and replication fork progression relate to prior discussion of euchromatin vs heterochromatin and how chromatin state influences replication timing.
Epigenetic inheritance via the histone code connects DNA replication to mechanisms of gene regulation and cell identity discussed previously.
Telomere biology ties into aging and cancer biology: replicative senescence as a tumor-suppressive mechanism and the role of telomerase in stem cells and certain cancers.

Understanding telomerase activity informs potential therapies for aging and cancer, including boosting telomerase in stem cells or inhibiting it in cancer cells.
The balance between preventing cancer (via replicative senescence) and tissue renewal (via telomerase activity in stem cells) has implications for regenerative medicine and aging research.

Origins per chromosome: multiple origins; not all firing simultaneously; Euchromatin origins replicated earlier.
Histone gene copies in humans: 20 copies per histone gene.
Telomere repeat length: approximately 10^3 repeats of the sequence GGG TTA.
Telomere shortening rate: 100\text{ to }200 base pairs per division.
3' overhang: extended by telomerase to prevent loss of genetic information.
Lagging strand synthesis: performed by DNA polymerase alpha (with primase); leading/other roles attributed to the replication machinery (as discussed in lectures).