Stable Expression of Genes and Proteins with Age.

<p>(A) Expression of mitochondrial genes was analyzed by RT-qPCR normalized to <i>Gapdh</i> and presented relative to 1 month old mice. (B) Expression levels of proteins were evaluated by western analyses using antibodies as specified in Experimental Procedures. The relative increase in COXI and ND6 with age is indicated. Figure shows mean with SD. **p<0.01, *p< 0.05.</p

RNA Error Frequency is Age-Independent in Controls but Elevated in Mutator Mice.

<p>(A) mtRNA errors were measured as described at the same 7 loci as for mtDNA from brains of young (1 months, n = 8) and old (18 months, n = 8). (B) mtRNA errors in heterozygous (mut/+, n = 3) and homozygous (mut/mut, n = 3) mice. Figures show mean with SD. p**<0.01 vs. 18 months, p*< 0.05 vs. 18 months.</p

Level of RNA Deletions is Age-Independent.

<p>Intentional capture of mtRNA deletions by PCR amplification of extended cDNA regions.</p

Recruitment of SWI/SNF chromatin remodeling factors to nuclear virus DNA replication foci.

<p>(<b>A</b>) SMARCB1 co-localizes with UL44 in HCMV-infected fibroblast cells harvested at 24, 48, and 72 hpi. (<b>B</b>) Co-localization of SMARCB1 and other essential factors of the SWI/SNF complex; BRG-1, BAF155, BAF170, in HCMV infected fibroblast cells harvested at 48 hpi. The cells were fixed and subjected to double-staining for UL44 (mouse Mab-UL44) and SMARCB1 (rabbit Pab-SMARCB1) and SMARCB1 (rabbit Pab-SMARCB1) and either BRG-1 (mouse Mab-BRG-1), BAF155 (mouse Mab BAF155), BAF170 (mouse Mab BAF170) for immunofluorescence microscopy. Secondary antibodies were used for staining UL44, BRG-1, BAF155, BAF170 in green (anti-mouse 488) and SMARCB1 in red (anti-rabbit 594), and the cells were further visualized by confocal microscopy. Co-localization was visualized by a merge of the two microscopic determinations, and counterstaining of the nuclei was achieved by the use of DAPI.</p

SMARCB1, UL114 and UL44 are associated with the chromatin and the nuclear matrix in HCMV infected fibroblast cells.

<p>Mock- and HCMV-infected fibroblast cells harvested at indicated time points were subjected to sub-nuclear fractionation to obtain whole chromatin fraction and core nuclear matrix. Proteins from equal cell equivalents from each fraction were analyzed by western blotting with the indicated antibodies.</p

Increased expression of the SWI/SNF core subunits in HCMV infected cells.

<p>(<b>A</b>) The expression of UL114, SMARCB1 and UL44 in nuclear extracts (20 µg) from mock and HCMV-infected fibroblast cells was analyzed at immediate –early (12 hpi), early (24 hpi) and late (48 and 72 hpi) times of infection by western blot. The western blots were analyzed by Thyphoon scanning. GAPDH was used as a loading control. (<b>B</b>) The expression of BRG1, BAF155 and BAF 170 in nuclear extracts (40 µg) from mock and HCMV-infected fibroblast cells was analyzed at late (72 hpi) time of infection by western blot. The western blots were analyzed by Thyphoon scanning. GAPDH was used as a loading control.</p

Site- and Age- Dependent Accumulation of Mutations in Brain mtDNA.

<p>(A) DNA from young (1 month, n = 8) and old (18 months, n = 8) mice were analyzed for mutation rate in 7 different sites in the mtDNA. DNA was isolated from whole brain. (B) DNA from heterozygous (mut/+, n = 3) and homozygous (mut/mut, n = 3) mutator mice (9 months) were analyzed in the same sites. Figures show mean with SD. **p<0.01, *p< 0.05.</p

SMARCB1 and UL114 interact <i>in vitro</i>.

<p>(<b>A</b>) <i>In vitro</i> binding analysis of HA-tagged Δ379-SMARCB1 and c-myc-tagged UL114 in ³⁵S-labeled proteins using the TNT coupled transcription/translation system. The proteins were transcribed and translated <i>in vitro</i> with ³⁵S-methionine in the translation mixture to generate radioactive labeled products from vectors pACT2-Δ379-SMARCB1 (HA-epitope) and pGBKT7-UL114 (c-myc epitope). The translated Δ379-SMARCB1-HA and UL114-c-myc were immunoprecipitated with either anti-HA or anti-c-myc-antibodies and eluted from the Protein G beads. Samples were subjected to 8% SDS-PAGE and PhosphoImaging. Lane 1: UL114-c-myc+c-myc antibody. Lane 2: Δ379-SMARCB1-HA+HA-antibody. Lane 3: Δ379-SMARCB1-HA+UL114-c-myc+HA-antibody. Lane 4: Δ379-SMARCB1-HA+UL114-c-myc+c-myc antibody. Lane 5: UL114-c-myc+HA-antibody. Lane 6: Δ379-SMARCB1-HA+c-myc-antibody. (^.......) indicates that samples were run on the same gel and (——) indicates that samples were run on a different gel. (<b>B</b>) and (<b>C</b>) GST pull-down assays to detect the interaction of SMARCB1 and UL114. (<b>B</b>) Purified GST-SMARCB1 or crude extract of <i>E. coli</i> cells over-expressing GST were incubated with purified UL114. The GST pull-down products were immunoblotted with anti-SMARCB1, anti-UL114 and anti-GST. Lane 1: GST-extract+UL114. Lane 2: GST-SMARCB1+UL114. Lane 3: purified GST-SMARCB1 (2 µg, 10% of input). Lane 4: Purified UL114 (1 µg, 5% of input). Note that spontaneous cleavage of GST occurred in the GST-SMARCB1 protein sample (Lanes 2 and 3). (<b>C</b>) Purified GST-SMARCB1 or crude extract of <i>E. coli</i> cells over-expressing GST were incubated with lysates of HCMV-infected cells. The GST pull-down products were immunoblotted with anti-SMARCB1, anti-UL114, anti-GST and anti-UL57. Lane 1: GST-extract+HCMV lysate. Lane 2: GST-SMARCB1+HCMV lysate. Lane 3: purified GST-SMARCB1 (2 µg, 10% of input). Lane 4: HCMV lysate (30 µg, 1% of input). Note that spontaneous cleavage of GST occurred in the GST-SMARCB1 protein sample (Lanes 2 and 3). The asterisks (*) on Lanes 1 and 4 indicates unspecific bands by the use of anti-SMARCB1.</p

Validation of Method.

<p>(A) Flow chart illustrating the procedure for detection of mtRNA integrity. (B) Comparison of RNA integrity in CoxI site obtained by using two different commercially available reverse transcriptases. (C) Estimation of errors introduced in the PCR amplication. The 12S rRNA region was amplified with increasing cycle number and 100 ng PCR product was either digested with TaqI or left untreated and subsequently analyzed for mutations. The mutation frequency is plotted as function of PCR cycle number. The figures show mean with SD from three independent experiments.</p

Interaction of SMARCB1 and UL44 in HCMV-infected fibroblast cells and with recombinant proteins.

<p>(<b>A</b>) Co-immunoprecipitation of endogenous SMARCB1 and UL44 in HCMV-infected fibroblast cells. Equal numbers of mock infected and HCMV infected fibroblast cells were lysed at 72 hpi, and the extracts were immunoprecipitated with anti-SMARCB1. Co-immunoprecipitated proteins were resolved electrophoretically and subjected to immunoblot analysis with anti-UL44 and anti-SMARCB1. Lane 1: Mock lysate immunoprecipitated with anti-SMARCB1. Lane 2: HCMV lysate immunoprecipitated with anti-SMARCB1. Lane 3: HCMV lysate immunoprecipitated with no antibody. Lanes 4 and 5, SMARCB1 and UL44 input in extracts (7 µg, 1% of the total in Lane 4 and 35 µg, 5% of the total in Lane 5). IgGHc: IgG heavy chain. (<b>B</b>) <i>In vitro</i> pull-down assay of GST-SMARCB1 and His₆-UL44. Purified GST-SMARCB1 or GST incubated with purified His₆-UL44 immobilized on magnetic His-tag Dynabeads. Samples were analyzed by SDS-PAGE and Coomassie blue staining. Lane 1: GST-SMARCB1+Dynabeads His-tag. Lane 2: His₆-UL44+Dynabeads His-tag. Lane 3: GST-SMARCB1+His₆-UL44+Dynabeads His-tag. Lane 4: His₆-UL44 (2 µg, 10% of input). Lane 5: GST-SMARCB1 (2 µg, 10% of input). Lane 6: GST+His₆-UL44+Dynabeads His-tag. Lane 7: GST (2 µg, 10% of input). Note that spontaneous cleavage of GST occurred in the GST-SMARCB1 protein sample (Lane 5). (^……) indicates that samples were run on the same gel.</p