Scaffold proteins are known to interact with multiple proteins involved in a signaling pathway, tethering them into complexes. In such pathways, they regulate signal transduction and help localize pathway components.

siRNA is a type of RNA interference, a regulatory mechanism used for regulation of proteins.

In combination with an Ago protein, the seed sequence of the siRNA, found in the 5' UTR, functions as a RISC complex that can find and bind to complimentary mRNA sequences and target them for silencing.

The use of an siRNA resistant MUS81 sequence in this experiment allows for both the knock-down and introduction of MUS81 to observe it's affects.

MUS81-EME1 is responsible for cleaving DNA at stalled and collapsed replication forks, triggering a DNA repair pathway. However, when knocked down, new DNA synthesis was not observed, because stalled forks were not cleaved and the pathway to fix them was not triggered.

Hypothesis/question: how late in S phase does replication of CFSs under stress occur?

Major findings: 1. Replicative-stress-induced DNA synthesis can occur after entry into mitosis, but only before prometaphase. 2. Mitotic DNA synthesis occurs primarily at CFSs.

Experiments: EdU incorporation to detect new DNA synthesis under three conditions: stress, cell cycle step, location on chromosomes.

Specific markers that indicate the location of CFSs in the genome.

Occurrence of DNA synthesis was tracked and visualized specifically in cells that had progressed into mitosis after being exposed to replicative stress (APH inhibitor).

Cells that had already moved to prometaphase did not show signs of mitotic DNA synthesis - later papers showed otherwise.

Tells us that some level of DNA replication is occurring in mitosis, as late as anaphase.

40% is questionable among scientists.

(A) thymidine amino acid, (B) 5-bromo-2'-deoxyuridine antibody (BrdU), and (C) 5-ethynyl-2'-deoxyuridine azide (EdU).

EdU is incorporated into the genome during DNA replication and allows for the identification of newly synthesized DNA via fluorescence.

"Why" the authors did these experiments.

Major finding

Major finding

MUS81-EME1 promotes the appearance of chromosome gaps or breaks at CFSs by cleaving stalled replication forks, triggering a DNA damage response that can lead to chromosomal instability.

MUS81-EME1 is a DNA endonuclease that is recruited during prophase of the mitosis. MUS81-EME1 is involved in resolving DNA structures, such as Holliday junctions, formed during genetic recombination. These structures need to be resolved correctly to ensure accurate chromosome segregation during mitosis.

Holliday junction: involves the exchange of DNA strands between two homologous chromosomes or sister chromatids. Resolving a Holliday junction means cleaving the junction to allow the strands to separate properly, ensuring accurate recombination and genetic diversity.

Cancers have high mutation rates, which are increased further under replication stress.

Stalled replications forks arising from CFSs can collapse and form DNA breaks (SSBs and DSBs). This causes the gaps or breaks on metaphase chromosomes.

Common fragile sites are regions of a genome that exhibit difficult-to-replicate sequences and secondary structures (generally resulting from AT-rich repeats). These regions are generally stable, but when a cell experiences replicative stress, they can become more difficult to replicate, leading to incomplete replication of CFSs that are carried through into mitosis [1,2].

CFSs are hotspots for chromosomal instability and rearrangements in cancers [2].

1. T. Glover. Common fragile sites. Cancer Lett 2006. Doi: 10.1016/j.canlet.2005.08.032.
2. Li, S., Wu, X. Common fragile sites: protection and repair. Cell Biosci 2020. https://doi.org/10.1186/s13578-020-00392.

Tumorigenesis: conversion of a healthy cell into cancer cells.

Oncogene activation disrupts canonical cellular pathways, leading to genome instability.

• secondary structures resulting from AT-rich sequences
• Transcription–replication conflicts: cellular machineries responsible for gene expression and genome duplication collide with each other on the same genomic location.
• Lack of replication origins

Replication is a highly regulated process designed to guarantee accurate duplication of the genome once per cell cycle. Any condition that compromises it is referred to as replication stress.

When cells experience replication stress, replication of CFSs are particularly impacted, leading to uncomplete replication when cells enter mitosis.

The recurrent formation of gaps and breaks at CFSs highlights their inherent instability and vulnerability to disruptions in DNA replication processes.

Cytogenetically defined gaps and breaks: gaps and breaks on chromosomes at CFSs are visually identifiable under a microscope using cytogenetic techniques.

In an effort to elucidate the mechanisms that regulate cardiac and smooth muscle genes, scientists performed a BLAST search to look for potential cardiac-specific genes that may be involved in regulation.

Discovery of myocardin

Proteins that assist in the activation of genes involved in muscle formation and function

SRF doesn ot appear to function only in the muscles, so it likely relies on the recruitment of myogenic accessroy factors to accomblish gene activation.

SRF binds to the serum response element (SRE) in the promoter region of target genes.

When certain cells are stimulated by growth factors or mitogens, some of their genes get turned on temporarily. These genes are generally regulated by a region called the serum response element (SRE).

Mitogen = A mitogen is a small bioactive protein or peptide that induces a cell to begin cell division, or enhances the rate of division (mitosis).

1 of the 20 new, previously unidentified sequences matched with a region of the gene MYOCD known as the 3' untranslated region (UTR)

BLAST (Basic Local Alignment Search Tool): * Compares sequences of nucleotides or proteins to find similarities * Wang et. al. compared sequences derived from RNA (expressed sequence tags) obtained from libraries of embryonic cardiac muscle cells with sequences already known and recorded in databases

Transcriptomics: * Techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. * Can provide insight into gene expression.

individuals who inherit only one mutated copy of the gene (heterozygotes) typically have around a 50% deficiency in the corresponding enzyme. However, this level of deficiency is usually not enough to cause the disease.

Individuals who were homozygous recessive would have the disease.