Strand Breaks

We further wanted to determine the level of DNA damage in ATII cells isolated from emphysema patients compared to control non-smokers or smokers. We analyzed phosphorylation of H2AX (γH2AX), which is a sensitive indicator of DSBs (REF), by Western blotting and immunohistofluorescence. We found significantly higher γH2AX phosphorylation in ATII cells obtained from smokers in comparison with non-smokers (p<0.05; Figs. 2a, b). Interestingly, ATII cells isolated from emphysema patients have lower expression of γH2AX phosphorylation in comparison with smokers. We also determined γH2AX phosphorylation in lung tissue sections by immunohistofluorescence using SP-A as a marker of ATII cells. We obtained similar results showing higher γH2AX florescence intensity in ATII cells obtained from smokers in comparison with control non-smokers and emphysema (p< 0.05; Figs. 2c, d). Although, ATII cells isolated from emphysema patients have higher ROS levels (Figs. 1a, b), they don’t show a significant increase in γH2AX phosphorylation. This may suggest the impairment of DSBs repair signaling leading to lack of the DNA damage repair in these cells.

During prophase the spindle fibers are developed and moving towards opposite poles. During metaphase the chromatids aligns at the plane of the cell equator. During anaphase, the paired sister chromatids are separated and each move towards one pole, at this point the cohesin at the centromere are removed. Cohesin, a protein complex, plays an essential role during this process. The core subunits of the cohesin complex are: Smc 1 and 3 (structural maintenance of chromosome (5)), Rad21, and Scc 2-4 (adherin factor) which form a ring-like structure that performs cohesion on the sister chromatids in S phase and is maintained until metaphase (4). The Smc proteins contain ATPase (ABC type) at the terminal domains separated by a coiled coil domains and hinge domains which allows for interaction between the N- and C-terminals which are bridge by a heterodimer (Rad21 and Scc3 complex) (1). Cohesion process requires the Smc proteins to have the hinge domain, and ATPase activity in order for cohesin to associate with chromatin during the G1-phase (1,4). The Scc2-4 complex is required to load the cohesin complex onto chromatin (6). Scc complexes are used to mediate protein-protein interactions essential for cell division. Depletion in the protein results in severe premature sister chromatid separation (PCS) that will be discussed later (7). Once cohesion is established, Pds5 complex serves to maintain the sister chromatid structure through the G2-phase

In the synapsis step, the Rad51-ssDNA filament (presynaptic filament) performs homology search and DNA strand invasion on a homologous region of another duplex, which results in the formation of a displacement loop (D-loop) [40]. In the post-synapsis stage of HR the invading 3′ end of the D-loop primes DNA synthesis by a DNA polymerase, which extends the D-loop enabling the second 3’ single strand terminal end of the DSB to base-pair with it - a process that is called second end capture. Once captured the second end can itself prime DNA synthesis, and the result of these “DNA transactions” is the covalent linkage of the recombining DNA molecules via two HJs. Depending on how the HJs are processed to form mature recombinant products three sub-pathways of HR are distinguished. In the canonical DSBR model the two HJs (doubleHJ/dHJ) can move along the DNA by a process called branch migration, which extends or limits the region of DNA heteroduplex formed by strand exchange catalyzed in the second step. Subsequently, dHJ intermediate could be resolved by endonucleases such as the Mus81-Eme1/Mms4 complex, Slx1-Slx4 and the GEN1/Yen1, resulting in the formation of either crossovers (CO) or non-crossover (NCO) recombinants depending on the orientation in which each HJ is cleaved

Apart from PTMs of the H3 nucleosomes, the centromere code is also unique because CENP-A nucleosomes also undergo PTMs and regulate centromere function (Figure 1). The centromeric histone H4 of CENP-A containing nucleosomes undergo monomethylation at Lysine 20 following deposition of CENP-A into centromeres[94, 95]. When present in the H3 nucleosome, H4K20me1 is associated with transcribed regions throughout the genome[96]. Targeting the H4K20me1-specific demethylase PHF8 to centromeres reduces centromeric level of H4K20me1 and results in reduced recruitment of CENPT and CENP-H[94]. It is not yet clear whether this conserved H4K20me1 is directly responsible for the recruitment of the kinetochore components, or if it is an indirect effect caused by reduced

In addition, it is also shown that γ-irradiation causes DNA lesions that are not well repaired in HGPS cells4. As the study shows, molecular details about how repair proteins and checkpoints are responsible for DNA repair mechanism are still unknown4. To unravel the complications in deficiency of DNA repair mechanisms and their relationship with H3K9me3, immunofluorescence staining and confocal microscopy were carried out and it was found that γ-H2AX/53BP1 foci were associated with regions enriched with H3K9me34. Moreover, to analyze the hypothesis that H3K9me3 poses a barrier for defective DNA repair, SUV39 was knocked down by siRNA and it was revealed that DNA damage foci decreased significantly which proved the hypothesis tested4. In addition to this hypothesis, it should be also proven if SUV39H1 levels directly contribute to deficiency of DNA repair and checkpoint response proteins in progeria. For this purpose, levels of SUV39H1mRNA in cells with ectopic progerin were analyzed and it was concluded that SUV39H1 levels were significantly increased in mouse embryonic fibroblasts (MEFs) in comparison to the wild type control4. Based on previous hypotheses, it can be implied that since H3K9me3 is mainly catalyzed by SUV39H1 methyltransferase, levels of H3K9me3 must be higher in Zmpste24 - / -

The mitochondrial pathway, also called intrinsic pathway, because it is initiated from inside the cell. Various stimuli such as growth factors withdrawal, DNA-damage, hypoxia, and oxidative stress can induce apoptosis through this cascade. These insults cause increasing permeability of the outer mitochondrial membrane and opening of the mitochondrial permeability transition (MPT) pore which is controlled by members of the Bcl-2 family proteins. This large family of proteins is defined by the presence of conserved Bcl-2 homology domains (BH1 to BH4). Up to 30 Bcl-2 family genes have been identified in mammals, which have either pro-apoptotic or anti-apoptotic functions. Some of the anti-apoptotic members include Bcl-2 itself, Bcl-XL, Bcl-w, BAG and Mcl-1 which possess all domains of BH1 to BH4. The pro-apoptotic family proteins can be divided into two subgroups: consists of Bak, Bax, and Bok with possess BH1 to BH3 domains, and Bad, Bid, Bik, BNIP3, Bim, Bmf, Blk, Hrk, Noxa, Puma, and Spike) that only possesses BH3 domain [Cory, 2002; Mund, 2003]. It is believe that BH3-only proteins interfere with the fine-tuned balance of homo- or hetero-oligomerization between pro-apoptotic multidomains (eg., Bax/Bak) and anti-apoptotic members (eg., Bcl-2/Bcl-XL) (Figure 3). In general, oligomers of Bak, Bax, and Bok induce PMT, either by forming channels by themselves or by interacting with components of the PMT [Antonsson, 2000]. Bad can also heterodimerize

Cdk1 is essential for mitosis and meiosis whereby in the absence of Cdk2, mitotic cell division can still occur (Santamaría et al., 2007; Berthet et al., 2013). Cdk1 is able to compensate the loss of the Cdk2 as it is found to be able to bind to cyclin E to promote the S phase entry (Aleem et al., 2005), even though it can cause a slight delay in the S phase. Cdk2 is known to be required in the germ cell development. This indicates that the loss of Cdk2 would results in sterility (Berthet et al., 2013).

Among the many types of DNA damage, one of the most severe is DNA double strand breaks (DSB). DNA DSBs occur when two complementary strands break simultaneously generating two strands which are not being held together by base-pairing and chromatin structure. Few known causes for DSB are ionizing radiation, chemical mutagens, reactive oxygen species and drugs which are used for chemotherapy (Goodsell, 2005). This breakage can cause chromosomal rearrangement such as translocation and inversion, consequentially resulting in detrimental phenotypic affects.

The transformation of somatic cells to cancerous cells is usually a slow and accumulative process, brought about by external sources causing mutations to the genetic material of the cells (DNA or RNA). Many intrinsic mechanisms, such as the mismatch repair system, are implemented to reverse

After DNA damage, due to the accumulation of mismatch repair, it causes genomic instability. Besides, the genetic information is changed. P53 can participate in DNA repair process, which itself has a DNA-binding domain nucleic acid endonuclease activity, resectable mismatch nucleotide, nucleotide binding and regulate endo repair factor XPB and XPD activity, affect its DNA recombination and

Since this is the only part of the histone protein which exists in an uncoiled state, it is subjected to far more modifications than the core proteins. Structurally, the H1 proteins exhibits a much longer C-terminal domain as compared to its N-terminal domain surrounding a highly conserved central globular domain. This C-terminal domain is arranged as a β hairpin motif associated with three α helices. The C-terminal is lined with Lysine residues with alternating Alanine residues. The amino acid constitution of the N and C terminals of this linker histone is detrimental for chromatin folding. The N-terminal region is composed of hydrophobic residues and the globular domain is made up of mostly basic amino acids; however the exact composition still remains to be characterized. Research has revealed the C-terminal domain by itself is able to bring about an efficient amount of chromatin fiber folding as the native H1 protein. Phosphorylation of this linker histone protein is the most studied post translational modification. Research has brought forward numerous examples of cross-talk found to be essential for regulation of this protein. Acetylation of Lysine at position 34 by HAT enzyme has been proven to be associated with activation of DNA

One such form of DNA damage includes DNA double stranded breaks (DSBs), a complex form of DNA damage that affects both strands of the double helix at the same point. These breaks are dangerous for the cell as neither strand can serve as a template for repair. Nonetheless, the cell can repair DSBs using several pathways, namely Non-Homologous End Joining (NHEJ), Microhomology-mediated End Joining (MMEJ), and Homologous

The gRNA directs the Cas9 endonuclease to the specific sites in the genome and causes a double-strand break (DSB). The host cells repair the DNA damage through the non-homologous end joining pathway and can introduce insertions and deletions in the target site in the process (Yu et al., 2017).

Proper gene expression is crucial for normal physiological development. Unfortunately, the structural and functional integrity of cellular DNA is constantly at risk by intrinsic and extrinsic factors, from mistakes in metabolic processes to radiation damage. DNA repair pathways are critical processes that address these offenses by maintaining the level genomic integrity necessary for accurate cellular division and function. These pathways involve the seamless integration of repair with processes that operate on DNA, such as replication and transcription [1]. However, the coordination of these various pathways in DNA repair and the mechanisms by which specific sites of damage are recognized remain poorly characterized.

Background: The Oocyte, a highly differentiated cell type, has the remarkable capacity to reprogram cells (from sperm to even somatic cells) to that of totipotent embryonic cells. Understanding how oocyte-derived factors contribute to developmental competence will not only impact the fields of stem cell biology/cellular reprogramming but also benefit to infertile patients directly. I have recently shown that Hira mediated histone variant H3.3 incorporation is involving the nucleosome assembly in the male genome to form a male pronucleus. Moreover, I demonstrated that maternal H3.3 is required for zygotic cleavage to 2-cells by regulating the function of RNA Polymerase I (ribosomal RNA, rRNA, transcription). This serendipity overturned a long-lasting dogma that transcription of the mouse zygotic genome is minor and not required for development. However, the molecular mechanisms remain to be elucidated. RRNAs are transcribed using rDNA gene array, which is an epigenetically regulated repeated gene locus. My overall hypothesis is that unique epigenetic regulatory mechanisms of ribosome RNA transcription occur during oogenesis is critical for zygote development. To prove this, I will continue explore how Hira complex and H3.3 regulate rDNA transcription. I will next explore the upstream processes of ribosome